Compositions and methods for controlling arthropod parasite and pest infestations

ABSTRACT

This application provides and discloses anti-parasitic, anti-pest or insecticidal nucleic acid molecules and their calmodulin target genes for the control of arthropod parasites and pests. This application further provides methods and compositions for the control and treatment of parasites and pests in  Apis mellifera  (honey bee) hives.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S.Provisional Application No. 61/899,772, filed Nov. 4, 2013, which isherein incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the Sequence Listing is filed with thisapplication by electronic submission and is incorporated into thisapplication by reference in its entirety. The Sequence Listing iscontained in the file created on Mar. 2, 2015, having the file nameP34094US01_SEQ.txt, and is 64,071 bytes in size (as measured in theMS-Windows® operating system).

FIELD OF THE DISCLOSURE

Methods and compositions for controlling parasite and pest infestationsof arthropods are provided. Also provided are methods and compositionsfor controlling Varroa mite infestation in bees.

BACKGROUND

Arthropods of various species are increasingly cultured on a commercialscale. Insects and their grubs are nutritious and are eaten both raw andcooked in many cultures. Crustaceans such as crabs, lobsters, crayfish,shrimp and prawns are farmed on a large commercial scale and are animportant part of the human diet. In addition to the culture ofarthropod species for food, arthropods are also cultured as part of pestmanagement strategies, including for the biological control of otherarthropods, for example the culture parasitic wasps for the control ofroaches and fire ants. Arthropods may also serve as the source of rawmaterials such as dyes, drugs, medicines, and antibiotics. Growing withthe increasing importance of arthropod culture, are various pests andparasites that destroy the arthropod colonies or greatly reduce theyields of products obtained from arthropod culture. Accordingly, thereis an increasing need for methods to control arthropod pests andparasites.

Among the most important species of cultured arthropods is the honeybee. Honey bees, Apis mellifera, are required for the effectivepollination of crops and are therefore critical to world agriculture.Honey bees also produce economically important products, including honeyand bees wax. Honey bees are susceptible to a number of parasites andpathogens, including the ectoparasitic mite, Varroa destructor.

Varroa (Varroa destructor) mites are the number one parasite of managedhoney bees (Apis mellifera) and the biggest global threat to commercialbeekeeping (Rosenkranz et al. 2010). An adult mite typically enters theworker and drone brood cells before they are capped, primed by honeybeebrood pheromone. The mite submerges into the brood food that the beesput inside the cell in anticipation of capping, most probably to avoidbeing recognized and removed by nurse bees. Following capping of thebrood cells by the nurse bees, the mite adheres to the larva and startsto ingest bee larval hemolymph. This process primes oogenesis in themites, and is followed several days later in laying of male and femaleeggs. Eventually, the adult Varroa exit the cell and cling onto theemerging bees. Varroa directly damages the honeybees in multiple ways,most notably by draining resources, adversely affecting the innate honeybee immune system, and by being a very effective vector of viruses (DiPrisco et al. 2011), some of which are known to replicate in the mite,thus dramatically increasing the viral load.

A safe, efficacious and long-lasting solution to the Varroa problem isan ongoing challenge that has yet to be met. Currently, beekeepers use aplethora of methods to control Varroa levels that include variouschemical miticides, most of which have lost efficacy and are toxicand/or leave residues in wax and honey. Other methods includeapplication of oxalic or formic acid, monoterpenes (thymol) and avariety of other management practices, with highly variable outcomes,including toxicity to the treated colonies. Breeding of bees forresistance to Varroa, such as selection for Hygienic behavior whichresults in the removal of infested brood, has provided a limitedpractical success.

Colony Collapse Disorder (CCD) of honeybees is threatening to annihilateU.S. and world agriculture. Indeed, in the recent outbreak of CCD in theU.S in the winter of 2006-2007, an estimated 25% or more of the 2.4million honeybee hives were lost because of CCD. An estimated 23% ofbeekeeping operations in the United States suffered from CCD over thewinter of 2006-2007, affecting an average of 45% of the beekeepersoperations. In the winter of 2007-2008, the CCD action group of theUSDA-ARS estimated that a total of 36% of all hives from commercialoperations were destroyed by CCD.

CCD is characterized by the rapid loss from a colony of its adult beepopulation, with dead adult bees usually found at a distance from thecolony. At the final stages of collapse, a queen is attended only by afew newly emerged adult bees. Collapsed colonies often have considerablecapped brood and food reserves. The phenomenon of CCD was first reportedin 2006; however, beekeepers noted unique colony declines consistentwith CCD as early as 2004. Various factors such as mites and infectiousagents, weather patterns, electromagnetic (cellular antennas) radiation,pesticides, poor nutrition and stress have been postulated as causes. Todate, control of CCD has focused on Varroa mite control, sanitation andremoval of affected hives, treating for opportunistic infections (suchas Nosema) and improved nutrition. No effective preventative measureshave been developed to date.

Varroa mites parasitize pupae and adult bees and reproduce in the pupalbrood cells. The mites use their mouths to puncture the exoskeleton andfeed on the bee's hemolymph. These wound sites in the exoskeleton harborbacterial infections, such as Melissococcus pluton, which causesEuropean foulbrood. In addition, to their parasitic effects, Varroamites are suspected of acting as vectors for a number of honey beepathogens, including deformed wing virus (DWV), Kashmir bee virus (KBV),acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), andmay weaken the immune systems of their hosts, leaving them vulnerable toinfections. If left untreated Varroa infestations typically result incolony-level mortality.

Current methods of treating Varroa infestations are proving to beineffective as the mites develop resistance to existing miticides. Inaddition, the use of such miticides may introduce injurious chemicalsinto honey that is intended for human consumption.

SUMMARY OF THE INVENTION

The present disclosure provides for, and includes, selective insecticidecompositions comprising an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having a sequence that is essentiallycomplementary or essentially identical to a region of a calmodulin genesequence or an RNA transcribed therefrom. In some aspects, thecomposition further comprises an excipient.

In one aspect, the nucleic acid molecule in the selective insecticidecomposition is a dsRNA. In some aspects, the dsRNA is an siRNA.

In one aspect, the calmodulin gene sequence has at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selectedfrom SEQ ID NOs:1-4, 6, 23, 26-35, and 69-89. In some aspects, thecalmodulin gene sequence comprises at least 18 contiguous nucleotides ofa sequence selected from SEQ ID NOs: 1-4, 6, 23, 26-35, and 69-89.

In one aspect, the selective insecticide composition further comprisesone or more anti-parasitic, anti-pest or insecticidal nucleic acidmolecules that are essentially complementary or essentially identical toa first region of a calmodulin gene sequence. In some aspects, the oneor more nucleic acid molecules comprise a second nucleic acid sequencecomplementary to a second region of a calmodulin gene sequence.

In one aspect, the selective insecticide composition is bee-ingestible,bee-absorbable, mite-ingestible, or mite-absorbable.

In one aspect, the expedient is selected from the group consisting ofprotein, pollen, carbohydrate, polymer, liquid solvent, sugar syrup,sugar solid, and semi-solid feed. In some aspects, the liquid solvent isselected from the group consisting of sucrose solution and corn syrupsolution. In some aspects, the protein is selected from the groupconsisting of pollen and soy protein. In another aspect, the excipientis a solid selected from sugar, a sugar substitute, or a sugarsupplement. In some aspects, the sugar solid comprises sugarmicroparticles impregnated with a dsRNA nucleic acid sequence.

In one aspect, the instant application discloses bee-ingestiblecompositions comprising a bee feed and a nucleic acid molecule having asequence that is essentially identical or essentially complementary toone or more regions of a calmodulin gene sequence, or an RNA transcribedtherefrom. In some aspects, the bee feed comprises a bee food selectedfrom the group consisting of corn syrup, a pollen substitute, pollen, apollen patty, and a fondant. In some aspects, the bee feed furthercomprises one or more of a mineral salt, an essential oil, BrewersYeast, yeast extract, trehalose, tryptone, dry milk, lecithin, andVitamin C. Examples of essential oils include, but are not limited to,wintergreen oil, spearmint oil, peppermint oil, lemongrass oil and teatree oil.

In another aspect, the instant application discloses a nucleic acidconstruct comprising an anti-parasitic, anti-pest or insecticidalnucleic acid sequence that is essentially identical or complementary toa region of a calmodulin gene sequence, or an RNA transcribed therefrom,operably linked to a promoter sequence functional in a host cell andcapable of producing a dsRNA when introduced into said host cell. Insome aspects, the nucleic acid construct further comprises at least oneregulatory element selected from the group consisting of translationleader sequences, introns, enhancers, stem-loop structures, repressorbinding sequences, termination sequences, pausing sequences, andpolyadenylation recognition sequences. In some aspects, the host cell isa bacterial or yeast cell.

In another aspect, the instant application discloses a method ofproviding a composition to a honeybee, comprising providing the bee aneffective amount of a composition comprising an anti-parasitic,anti-pest or insecticidal nucleic acid that is essentially identical oressentially complementary to one or more regions of a calmodulin genesequence, or an RNA transcribed therefrom, whereby the nucleic acid ispresent in honeybee tissue.

In another aspect, the instant application discloses a method oftreating or preventing disease in a honeybee colony, comprisingproviding an effective amount of a composition comprising ananti-parasitic, anti-pest or insecticidal nucleic acid that isessentially identical or essentially complementary to one or moreregions of a calmodulin gene sequence to a honeybee whereby the nucleicacid is present in honeybee tissue. In some aspects, the calmodulin genesequence is a Varroa destructor calmodulin gene sequence.

In another aspect, the instant application discloses a method ofreducing parasitation of a bee by Varroa destructor, comprisingproviding the bee an effective amount of an anti-parasitic, anti-pest orinsecticidal nucleic acid composition, wherein the nucleic acid isessentially identical or essentially complementary to one or moreregions of a Varroa destructor calmodulin gene sequence, or an RNAtranscribed therefrom, thereby reducing the parasitation of the bee byVarroa destructor.

In another aspect, the instant application discloses a method ofreducing the parasite load of a honeybee hive, comprising providing saidhive an effective amount of an anti-parasitic, anti-pest or insecticidalnucleic acid that is essentially identical or essentially complementaryto one or more regions of a parasite calmodulin gene sequence, or an RNAtranscribed therefrom, whereby the parasite load of said hive isreduced.

In another aspect, the instant application discloses a method ofselectively treating an arthropod species for parasites, comprisingdelivering an effective amount of an anti-parasitic, anti-pest orinsecticidal nucleic acid that is essentially identical or essentiallycomplementary to one or more regions of a parasite calmodulin genesequence, or an RNA transcribed therefrom, to an arthropod species.

In another aspect, the instant application provides for, and discloses amethod of treating or preventing Colony Collapse Disorder in a honeybeecolony, comprising providing an effective amount of a composition to ahoneybee colony comprising an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having a sequence that is essentially identical toor essentially complementary to one or more regions of a Varroadestructor calmodulin gene sequence whereby the level of Varroadestructor infestation is reduced or prevented.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a phylogenetic tree for Calmodulin (CAM) genes fromdifferent species. The number immediately preceding the species namecorresponds to a Sequence Identification Number (SEQ ID NO).

FIG. 2 presents the survival rate of mites exposed to a nucleic acid ofSEQ ID NO: 3 (CAM373) in a direct feeding bioassay at 3 day posttreatment relative to a non treated control (CNTR) or a non-specificsequence (SCRAM, SEQ ID NO: 5).

FIG. 3 Panel A presents a gene expression analysis at five day posttreatment with a nucleic acid of SEQ ID NO: 3 (CAM373) or SEQ ID NO: 4(CAM186) relative to controls. Panel B shows the survival rate of mitesexposed to nucleic acids of SEQ ID NOS: 3 (CAM373) and 4 (CAM186)relative to controls.

FIG. 4 presents a mite load/100 bees of treated hives relative tountreated controls over a distinct time period.

FIG. 5 presents the % survival of mites treated with SEQ ID NO: 3, SEQID NO: 88 or SEQ ID NO: 89 relative to untreated (NTC) at Day 5 (D %) orDay 6 (D6) post-treatment.

FIG. 6 presents the % survival of mites treated with SEQ ID NO: 3 or amixture of SEQ ID NO: 88 and SEQ ID NO: 89 relative to untreated (NTC)at Day 5 (5), Day 6 (6) and Day 7 (7).

FIG. 7 presents the Varroa mite load/100 bees of treated hives relativeto untreated controls over a 17 week time period. The leftmost barsrepresent hives treated with the non-specific sequence (SCRAM, SEQ IDNO: 5), the middle bars are hives left untreated, and the rightmost barare hives treated with SEQ ID NO: 3 (CAM 373).

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms as used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. One skilled in the art will recognize many methods can be usedin the practice of the present disclosure. Indeed, the presentdisclosure is in no way limited to the methods and materials described.Any references cited herein are incorporated by reference in theirentireties. For purposes of the present disclosure, the following termsare defined below.

It is understood that any Sequence Identification Number (SEQ ID NO)disclosed in the instant application can refer to either a DNA sequenceor a RNA sequence, depending on the context where that SEQ ID NO ismentioned, even if that SEQ ID NO is expressed only in a DNA sequenceformat or a RNA sequence format. For example, SEQ ID NO: 1 is expressedin a DNA sequence format (e.g., reciting T for thymine), but it canrefer to either a DNA sequence that corresponds to a mature Varroadestructor calmodulin nucleic acid sequence, or the RNA sequence of amature Varroa destructor calmodulin molecule nucleic acid sequence.Similarly, though SEQ ID NO: 3 is expressed in a RNA sequence format(e.g., reciting U for uracil), depending on the actual type of moleculebeing described, SEQ ID NO: 3 can refer to either the sequence of a RNAmolecule comprising a dsRNA, or the sequence of a DNA molecule thatcorresponds to the RNA sequence shown. In any event, both DNA and RNAmolecules having the sequences disclosed with any substitutes areenvisioned.

As used herein the term “about” refers to ±10%.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

As used herein, “essentially identical” or “essentially complementary”refers to a nucleic acid (or at least one strand of a double-strandednucleic acid or portion thereof, or a portion of a single strand nucleicacid) that hybridizes under physiological conditions to the endogenousgene, an RNA transcribed therefrom, or a fragment thereof, to effectregulation or suppression of the endogenous gene. For example, in someaspects, a nucleic acid has 100 percent sequence identity or at leastabout 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99 percent sequence identity when compared to a region of 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more contiguousnucleotides in the target gene or RNA transcribed from the target gene.In some aspects, a nucleic acid has 100 percent sequence complementarityor at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, or 99 percent sequence complementarity when compared to aregion of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60or more contiguous nucleotides in the target gene or RNA transcribedfrom the target gene. In some aspects, a nucleic acid has 100 percentsequence identity with or complementarity to one allele or one familymember of a given target gene (coding or non-coding sequence of a gene).In some aspects, a nucleic acid has at least about 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequenceidentity with or complementarity to multiple alleles or family membersof a given target gene. In some aspects, a nucleic acid has 100 percentsequence identity with or complementarity to multiple alleles or familymembers of a given target gene.

In some aspects, the nucleic acid is essentially identical oressentially complementary to at least about 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70 or more contiguous nucleotides of an endogenouscalmodulin gene of a targeted pest, or an RNA transcribed therefrom. Thenucleic acid may be a single-stranded DNA, a single-stranded RNA, adouble-stranded RNA, a double-stranded DNA, or a double-stranded DNA/RNAhybrid. In some aspects, the calmodulin gene sequence is a Varroadestructor calmodulin gene sequence. In an aspect, the calmodulin genesequence is a calmodulin gene sequence selected from SEQ ID NO: 1. In anaspect, the calmodulin gene sequence is a calmodulin gene sequenceselected from SEQ ID NO: 2. In an aspect, the calmodulin gene sequenceis a calmodulin gene sequence selected from SEQ ID NO: 3. In an aspect,the calmodulin gene sequence is a calmodulin gene sequence selected fromSEQ ID NO: 4. In an aspect, the calmodulin gene sequence is a calmodulingene sequence selected from SEQ ID NO: 69. In an aspect, the calmodulingene sequence is a calmodulin gene sequence selected from SEQ ID NO: 70.In an aspect, the calmodulin gene sequence is a calmodulin gene sequenceselected from SEQ ID NOs: 71-87. In an aspect, the calmodulin genesequence is a calmodulin gene sequence selected from SEQ ID NO: 88. Inan aspect, the calmodulin gene sequence is a calmodulin gene sequenceselected from SEQ ID NO: 89.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition. In an aspect according to thepresent disclosure, a composition may be used to treat an organism orcolony of organisms for the effects of parasitation. In an aspect, anucleic acid composition may be used to treat a host organism or colonyfor parasites. In an aspect, the host organism is a bee and the parasiteis the mite, Varroa destructor.

As used herein, the phrase “RNA silencing” refers to a group ofregulatory mechanisms (e.g. RNA interference (RNAi), transcriptionalgene silencing (TGS), post-transcriptional gene silencing (PTGS),quelling, co-suppression, and translational repression) mediated by RNAmolecules which result in the inhibition or “silencing” of theexpression of a corresponding protein-coding gene or bee pathogen RNAsequence. RNA silencing has been observed in many types of organisms,including plants, animals, and fungi. In aspects according the presentdisclosure, nucleic acid compositions provide for RNA silencing. Incertain aspects, the nucleic acid compositions provide for RNA silencingand mortality in a parasite.

As used herein, the term “RNA silencing agent” refers to a nucleic acidwhich is capable of inhibiting or “silencing” the expression of a targetgene. In certain aspects, the RNA silencing agent is capable ofpreventing complete processing (e.g., the full translation and/orexpression) of an mRNA molecule through a post-transcriptional silencingmechanism. RNA silencing agents can be single- or double-stranded RNA orsingle- or double-stranded DNA or double-stranded DNA/RNA hybrids ormodified analogues thereof. In some aspects, the RNA silencing agentsare selected from the group consisting of (a) a single-stranded RNAmolecule (ssRNA), (b) a ssRNA molecule that self-hybridizes to form adouble-stranded RNA molecule, (c) a double-stranded RNA molecule(dsRNA), (d) a single-stranded DNA molecule (ssDNA), (e) a ssDNAmolecule that self-hybridizes to form a double-stranded DNA molecule,and (f) a single-stranded DNA molecule including a modified Pol III genethat is transcribed to an RNA molecule, (g) a double-stranded DNAmolecule (dsDNA), (h) a double-stranded DNA molecule including amodified Pol III promoter that is transcribed to an RNA molecule, (i) adouble-stranded, hybridized RNA/DNA molecule, or combinations thereof.In some aspects these polynucleotides include chemically modifiednucleotides or non-canonical nucleotides. In some aspects, the RNAsilencing agents are noncoding RNA molecules, for example RNA duplexescomprising paired strands, as well as precursor RNAs from which suchsmall non-coding RNAs can be generated. In some aspects, the RNAsilencing agents are dsRNAs such as siRNAs, miRNAs and shRNAs. In oneaspect, the RNA silencing agent is capable of inducing RNA interference.In another aspect, the RNA silencing agent is capable of mediatingtranslational repression. In an aspect, the RNA silencing agent iscapable of inhibiting the expression of a calmodulin gene. In anotheraspect, the RNA silencing agent is capable of being used in methods toinhibit the expression of a target gene and thereby kill a targetorganism. In certain aspects, the target gene is a calmodulin gene andthe target organism is Varroa destructor.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by small RNAs.The corresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. While not being limited to anyparticular theory, the process of post-transcriptional gene silencing isthought to be an evolutionarily-conserved cellular defense mechanismused to prevent the expression of foreign genes and is commonly sharedby diverse flora and phyla. Such protection from foreign gene expressionmay have evolved in response to the production of double-stranded RNAs(dsRNAs) derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. In aspects according to the present disclosure, a nucleic acidcomposition results in RNA interference in a target organism. In certainaspects, the nucleic acid composition results in RNA interference inVarroa destructor when present in the host organism, the bee. Accordingto aspects of the present disclosure, a selective insecticide may causeRNA interference in the targeted organism, while having no RNAinterference activity in non-target organisms.

As used herein, “small RNA” refers to any RNA molecule that is at least15 base pairs in length, generally 15-30 nucleotides long, preferably20-24 nucleotides long. In aspects according to the present disclosure,a “small RNA” is greater than 50 base pairs in length. In an aspect, thesmall RNA is greater than 50 base pairs in length but less than about500 base pairs. In an aspect, the small RNA is greater than 100 basepairs in length but less than about 500 base pairs. In an aspect, thesmall RNA is greater than 200 base pairs in length but less than about500 base pairs. A small RNA can be either double-stranded orsingle-stranded. Small RNA includes, without limitation, miRNA(microRNA), ta-siRNA (trans activating siRNA), siRNA, activating RNA(RNAa), nat-siRNA (natural anti-sense siRNA), hc-siRNA (heterochromaticsiRNA), cis-acting siRNA, 1miRNA (long miRNA), 1siRNA (long siRNA) andeasiRNA (epigenetically activated siRNA) and their respectiveprecursors. In some embodiments, siRNA molecules of the disclosure aremiRNA molecules, to-siRNA molecules and RNAa molecules and theirrespective precursors. A small RNA may be processed in vivo by anorganism to an active form. According to aspects of the presentdisclosure, a selective insecticide may be a small RNA.

In aspects according to the present disclosure, a small RNA is provideddirectly in a composition. In other aspects, a small RNA is produced byin vivo by an organism from either a DNA or an RNA precursor. In someaspects, the small RNA is produced as a product of a transgene in anorganism, for example a yeast or bacterial cell. In certain aspects, asmall RNA produced as a product of a transgene is produced as aprecursor that is processed in vivo after ingestion or absorption by anorganism. In other aspects, a small RNA produced as a product of atransgene is produced as a precursor that is processed in vivo afteringestion or absorption by an organism.

In some aspects, the RNA silencing agent may be an artificial microRNA.As used herein, an “artificial microRNA” (amiRNA) is a type of miRNAwhich is derived by replacing native miRNA duplexes from a natural miRNAprecursor. Generally, an artificial miRNA is a non-naturally-existingmiRNA molecule produced from a pre-miRNA molecule scaffold engineered byexchanging a miRNA sequence of a naturally-existing pre-miRNA moleculefor a sequence of interest which corresponds to the sequence of anartificial miRNA. In aspects according to the present disclosure anucleic acid composition may be an amiRNA composition.

Various studies demonstrate that long dsRNAs can be used to silence geneexpression without inducing the stress response or causing significantoff-target effects—see for example (Strat et al., Nucleic AcidsResearch, 2006, Vol. 34, No. 13 3803-3810; Bhargava A et al. Brain Res.Protoc. 2004; 13:115-125; Diallo M., et al., Oligonucleotides. 2003;13:381-392; Paddison P. J., et al., Proc. Natl Acad. Sci. USA. 2002;99:1443-1448; Tran N., et al., FEBS Lett. 2004; 573:127-134). Thepresent disclosure provides for, and includes, methods and compositionshaving long dsRNAs.

As used herein, with respect to a nucleic acid sequence, nucleic acidmolecule, or a gene, the term “natural” or “native” means that therespective sequence or molecule is present in a wild-type organism, thathas not been genetically modified or manipulated by man. A small RNAmolecule naturally targeting a target gene means a small RNA moleculepresent in a wild-type organism, the cell has not been geneticallymodified or manipulated by man which is targeting a target genenaturally occurring in the respective organism.

As used herein, the terms “homology” and “identity” when used inrelation to nucleic acids, describe the degree of similarity between twoor more nucleotide sequences. The percentage of “sequence identity”between two sequences is determined by comparing two optimally alignedsequences over a comparison window, such that the portion of thesequence in the comparison window may comprise additions or deletions(gaps) as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity. A sequence that is identical at every position incomparison to a reference sequence is said to be identical to thereference sequence and vice-versa. An alignment of two or more sequencesmay be performed using any suitable computer program. For example, awidely used and accepted computer program for performing sequencealignments is CLUSTALW v1.6 (Thompson, et al. Nucl. Acids Res., 22:4673-4680, 1994).

As used herein, the terms “exogenous polynucleotide” and “exogenousnucleic acid molecule” relative to an organisms refer to a heterologousnucleic acid sequence which is not naturally expressed within thatorganism. An exogenous nucleic acid molecule may be introduced into anorganism in a stable or transient manner. An exogenous nucleic acidmolecule may comprise a nucleic acid sequence which is identical orpartially homologous to an endogenous nucleic acid sequence of theorganism or a pest or pathogen of that organism. In certain aspects, an“exogenous polynucleotide” and “exogenous nucleic acid molecule” mayrefer to a parasite nucleic acid sequence expressed or present in ahost, either transiently or stably. The present disclosure provides for,and includes, compositions comprising exogenous polynucleotides andexogenous nucleic acid molecules and methods for introducing them into atarget organism. In some aspects, the present disclosure provides for,and includes, compositions comprising exogenous polynucleotides andexogenous nucleic acid molecules and methods for introducing them into anon-target organism that is a host to the target organism.

As used herein, a “control organism” means an organism that does notcontain the recombinant DNA, small RNA, or other nucleic acid (e.g.,protein, miRNA, small RNA-resistant target mRNA, dsRNA, target mimic)that provides for control of a pest or parasite. Control organisms aregenerally from same species and of the same developmental stage which isgrown under the same growth conditions as the treated organism.Similarly, a “control colony” means a colony of organisms that do notcontain the recombinant DNA, small RNA, or other nucleic acid (e.g.,protein, miRNA, small RNA-resistant target mRNA, target mimic) thatprovides for control of a pest or parasite. Control colonies oforganisms are generally from same species and of the same developmentalstage which are grown under the same growth conditions as the treatedcolony of organisms. As a non-limiting example, a control organism couldbe a bee provided with a composition that does not contain a nucleicacid of the present disclosure. In another non-limiting example, acontrol organism could be a bee provided with a composition thatcontains a nucleic acid that does not act a an RNA silencer in either abee or a parasite, such as SEQ ID NO: 5.

As used herein, the terms “improving,” “improved,” “increasing,” and“increased” refer to at least about 2%, at least about 3%, at leastabout 4%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, or greater increase in an organism or colony population, inincreased productivity of an organism or colony (e.g., increased honeyproductions), increase growth rate of an organism or colony, orincreased reproductive rate as compared to a control organism or colony.The present disclosure provides for methods of improving the health ofan organism or colony by providing a selective insecticidal composition.

As used herein, “a reduction” of the level of an agent such as a proteinor mRNA means that the level is reduced relative to an organism orcolony lacking a nucleic acid capable of reducing the agent. Also asused herein, “a reduction” in reference to parasitation or parasiteload, means that the level is reduced relative to an organism or colonylacking a nucleic acid, such as a dsRNA molecule, capable of reducingthe viability, fecundity or number of the parasite. The presentdisclosure provides for, and includes, methods and compositions forreducing the level of a protein or mRNA and reducing the level or numberof parasites.

As used herein, the term “at least a partial reduction” of the level ofan agent, such as a protein or mRNA, means that the level is reduced atleast 25% relative to an organism or colony lacking a nucleic acid, suchas a dsRNA molecule, capable of reducing the agent. Also as used herein,“at least a partial reduction” in reference to parasitation or parasiteload, means that the level is reduced at least 25% relative to anorganism or colony lacking a nucleic acid, such as a dsRNA molecule,capable of reducing the viability, fecundity or number of the parasite.The present disclosure provides for, and includes, methods andcompositions for at least partially reducing the level of a protein ormRNA and at least partially reducing the level or number of parasites.

As used herein, “a substantial reduction” of the level of an agent suchas a protein or mRNA means that the level is reduced relative to anorganism or colony lacking a nucleic acid, such as a dsRNA molecule,capable of reducing the agent, where the reduction of the level of theagent is at least 75%. Also as used herein, “a substantial reduction” inreference to parasitation or parasite load, means that the level isreduced at least 75% relative to an organism or colony lacking a nucleicacid, such as a dsRNA molecule, capable of reducing the viability,fecundity or number of the parasite. The present disclosure providesfor, and includes, methods and compositions for substantially reducingthe level of a protein or mRNA and substantially reducing the level ornumber of parasites.

As used herein, “an effective elimination” of an agent such as a proteinor mRNA is relative to an organism or colony lacking a dsRNA moleculecapable of reducing the agent, where the reduction of the level of theagent is greater than 95%. An agent, such as a dsRNA molecule, ispreferably capable of providing at least a partial reduction, morepreferably a substantial reduction, or most preferably effectiveelimination of another agent such as a protein or mRNA, or a parasite,wherein the agent leaves the level of a second agent, or host organism,essentially unaffected, substantially unaffected, or partiallyunaffected. Also as used herein, “an effective elimination” in referenceto parasitation or parasite load, means that the level is reduced atleast 95% relative to an organism or colony lacking a nucleic acid, suchas a dsRNA molecule, capable of reducing the viability, fecundity ornumber of the parasite. The present disclosure provides for, andincludes, methods and compositions for the effective elimination of aprotein or mRNA and effectively eliminating parasites.

As used herein, the terms “suppress,” “repress,” and “downregulate” whenreferring to the expression or activity of a nucleic acid molecule in anorganism are used equivalently herein and mean that the level ofexpression or activity of the nucleic acid molecule in a cell of anorganism after applying a method of the present disclosure is lower thanits expression or activity in the cell of an organism before applyingthe method, or compared to a control organism lacking a nucleic acidmolecule of the disclosure. The present disclosure provides for, andincludes, methods and compositions for suppressing, repressing anddown-regulating the level of a protein or mRNA and suppressing,repressing and down-regulating the level or number of parasites.

The terms “suppressed,” “repressed” and “downregulated” as used hereinare synonymous and mean herein lower, preferably significantly lower,expression or activity of a targeted nucleic acid molecule. Also as usedherein, “suppressed,” “repressed” and “downregulated” in reference toparasitation or parasite load, means that the level of parasitation orparasite load is lower, preferably significantly lower, relative to anorganism or colony lacking a nucleic acid, such as a dsRNA molecule,capable of reducing the viability, fecundity or number of the parasite.The present disclosure provides for, and includes, methods andcompositions for suppressing, repressing and down-regulating theexpression or activity of a protein or mRNA and suppressing, repressingand down-regulating the activity of parasites.

As used herein, a “suppression,” “repression,” or “downregulation” ofthe level or activity of an agent such as a protein, mRNA, or RNA meansthat the level or activity is reduced relative to a substantiallyidentical cell, organism or colony grown under substantially identicalconditions, lacking a nucleic acid molecule of the disclosure, forexample, lacking the region complementary to at least a part of theprecursor molecule of a dsRNA or siRNA, the recombinant construct orrecombinant vector of the disclosure. As used herein, “suppression,”“repression,” or “downregulation” of the level or activity of an agent,such as, for example, a preRNA, mRNA, rRNA, tRNA, snoRNA, snRNAexpressed by the target gene, and/or of the protein product encoded byit, means that the amount is reduced by 10% or more, for example, 20% ormore, preferably 30% or more, more preferably 50% or more, even morepreferably 70% or more, most preferably 80% or more, for example, 90%,relative to a cell, organism or colony lacking a recombinant nucleicacid molecule of the disclosure. The present disclosure provides for,and includes, methods and compositions for suppression, repression anddownregulation of an agent such as a protein, mRNA, RNA, or parasitecompared to an untreated organism or colony.

As used herein, the term “arthropod” refers to both adult and pupa ofinvertebrate animals having an exoskeleton (external skeleton), asegmented body, and jointed appendages. Arthropods are members of thephylum Arthropoda and includes the insects, arachnids, and crustaceans.Arthropods according to the present disclosure, include but are notlimited to Apis mellifera, Apis cerana, Trigona minima, Halictidae,Bombus sp., fleas, flies, lice, ticks, mites, and beneficial insects.The present disclosure provides for, and includes, methods andcompositions for treating arthropods as either a host or as a parasiteor pest.

In an aspect, an arthropod may be an insect. In certain aspects, aninsect may be a bee. As used herein, the term “bee” refers to both anadult bee and pupal cells thereof. According to one aspect, the bee isin a hive. An adult bee is defined as any of several winged,hairy-bodied, usually stinging insects of the superfamily Apoidea in theorder Hymenoptera, including both solitary and social species andcharacterized by sucking and chewing mouthparts for gathering nectar andpollen. Examples of bee species include, but are not limited to, Apis,Bombus, Trigona, Osmia and the like. In one aspect, bees include, butare not limited to bumblebees (Bombus terrestris), honeybees (Apismellifera) (including foragers and hive bees) and Apis cerana. Thepresent disclosure provides for, and includes, methods and compositionsfor treating bees as a host for parasites, such as Varroa mites.

According to one aspect, a bee is part of a colony. The term “colony”refers to a population of bees comprising dozens to typically severaltens of thousands of bees that cooperate in nest building, foodcollection, and brood rearing. A colony normally has a single queen, theremainder of the bees being either “workers” (females) or “drones”(males). The social structure of the colony is maintained by the queenand workers and depends on an effective system of communication.Division of labor within the worker caste primarily depends on the ageof the bee but varies with the needs of the colony. Reproduction andcolony strength depend on the queen, the quantity of food stores, andthe size of the worker force. Honeybees can also be subdivided into thecategories of “hive bees”, usually for the first part of a workerslifetime, during which the “hive bee” performs tasks within the hive,and “forager bee”, during the latter part of the bee's lifetime, duringwhich the “forager” locates and collects pollen and nectar from outsidethe hive, and brings the nectar or pollen into the hive for consumptionand storage. The present disclosure provides for, and includes, methodsand compositions for treating insects colonies.

As used herein, the term “pest” refers to both adult and immature formsof an organism that is invasive or prolific, detrimental, troublesome,noxious, destructive, a nuisance to either plants or animals, orecosystems. A parasite is a type of pest. It is possible for an organismto be a pest in one setting but beneficial, domesticated, or acceptablein another.

As used herein, the term “parasite” refers to both adult and immatureforms of organisms that directly benefit at the expense of another,host, organism, for example by feeding on the blood or fluids of thehost, living intracellularly in a host organism cell, or living within abody of a host organism. Parasites include organisms that are animals,fungi, bacterial or plants and are identified by their negative ordetrimental interaction with a host. In some aspects, a parasite as usedherein may in turn serve as a host to a second parasite. In someaspects, a parasite and host may be of the same type of organism (e.g.,an arthropod host and an arthropod parasite). Parasites include, but arenot limited to, Acari (ticks, mites), Hippoboscoidea (flies),Ichneumonoidea (parasitic wasps), Oestridae (bot flies), Phthiraptera(lice), Siphonaptera (fleas), Tantulocarida, Pea crab, and Sacculina. Asused herein, a pest may include both parasitic and non-parasitic lifestages. The present disclosure provides for, and includes, methods andcompositions for treating parasites. In an aspect, the parasite may beVarroa destructor.

As provided for, and included, in the present disclosure, parasitesand/or pests include Varroa destructor, Ixodes scapularis, Solenopsisinvicta, Tetranychus urticae, Aedes aegypti, Culex quinquefasciatus,Acyrthosiphon pisum, and Pediculus humanus. In aspects according to thepresent disclosure, selective insecticides may be selective for Varroadestructor, Ixodes scapularis, Solenopsis invicta, Tetranychus urticae,Aedes aegypti, Culex quinquefasciatus, Acyrthosiphon pisum, andPediculus humanus and inactive, or significantly less active, against anon-target organisms, such as the host organism.

As used herein, the term “excipient” refers to any inactive substance ina formulation having an active ingredient such as an anti-parasitic,anti-pest or insecticidal nucleic acid, including without limitationdsRNA, small RNAs, miRNAs and antisense RNAs. In some embodiments, anexcipient includes substances that may provide additional functionalityto a composition that is distinct to the anti-parasitic, anti-pest, orinsecticidal nucleic acids. Excipient functions include, but are notlimited to “bulking agents,” “fillers,” “diluents,” and “carriers.”Bulking up allows convenient and accurate dispensation of compositionsof the present disclosure. Excipients can also serve to facilitateingestion of the compositions by organisms and include variouscarbohydrates, proteins, fatty acids, pollens, and pollen substitutes.Excipients can also serve to facilitate absorption of compositions byorganisms an include, for example, both aqueous and non-aqueoussolutions of active ingredients. Non-limiting examples of excipientsinclude corn syrup, sugar syrup, sugar solid, sugar semi-solids, pollen,soy protein, pollen and protein mixtures. Excipients may furthercomprise attractants, buffers and nutrient supplements. Compositions ofthe present disclosure may be coated with, encapsulated in, dissolvedin, mixed with, or otherwise combined with an excipient. As used herein,the term excipient may refer to a mixture of inactive substances.

This application provides and discloses anti-parasitic, anti-pest orinsecticidal nucleic acid molecules that are substantially homologous orcomplementary to a polynucleotide sequence of a calmodulin target geneor an RNA expressed from the calmodulin target gene or a fragmentthereof and functions to suppress the expression of the calmodulintarget gene or produce a knock-down phenotype. The anti-parasitic,anti-pest or insecticidal nucleic acid molecules are capable ofinhibiting or “silencing” the expression of a calmodulin target gene.These nucleic acid molecules are generally described in relation totheir “target sequence.” In some embodiments, the target sequence isselected from SEQ ID NOs. 1, 2 and 6-77. The anti-parasitic, anti-pestor insecticidal nucleic acid molecules may be single-stranded DNA(ssDNA), single-stranded RNA (ssRNA), double-stranded RNA (dsRNA),double-stranded DNA (dsDNA), or double-stranded DNA/RNA hybrids. Thenucleic acid molecules may comprise naturally-occurring nucleotides,modified nucleotides, nucleotide analogues or any combination thereof.In some embodiments, a anti-parasitic, anti-pest or insecticidal nucleicacid molecule may be incorporated within a larger polynucleotide, forexample in a pri-miRNA molecule. In some embodiments, a anti-parasitic,anti-pest or insecticidal nucleic acid molecule may be processed into asmall interfering RNA (siRNA). In some embodiments, nucleic acidmolecules are provided or disclosed that are selectivelyanti-parasitical or miticidal, and methods of modulating expression oractivity of their target genes to reduce or eliminate parasites from acolony or population.

In aspects according to the present disclosure, a anti-parasitic,anti-pest or insecticidal nucleic acid moleculecomprises a nucleotidesequence having at least 80%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or99% sequence identity to a sequence or a portion of a sequence selectedfrom the group consisting of SEQ ID NOs: 1 to 89. In certain aspects,the nucleic acid molecule is sleeted from the group consisting of ssDNA,ssRNA, dsRNA, dsDNA, or DNA/RNA hybrids. Several embodiments relate to adsRNA comprising a nucleotide sequence having at least 80%, 85%, 88%,90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence ora portion of a sequence selected from the group consisting of SEQ IDNOs: 1 to 89. In another aspect, a DNA encoding at least one nucleicacid, such as a ssRNA or dsRNA, comprises a nucleotide sequence or aportion of a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 1 to 89, or having at least 80%, 85%, 88%, 90%, 92%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NOs: 1 to 89 or aportion thereof is provided. In yet another aspect, a recombinant DNAencoding at least one nucleic acid, such as a ssRNA or dsRNA, comprisesa nucleotide sequence or a portion of a nucleotide sequence selectedfrom the group consisting of SEQ ID NOs: 1 to 89, a heterologouspromoter and a transcription terminator sequence are provided. Inanother aspect, the present disclosure provides a recombinant DNAencoding at least one nucleic acid, such as a ssRNA or dsRNA, thatcomprises a nucleotide sequence having at least 80%, 85%, 88%, 90%, 92%,95%, 96%, 97%, 98%, or 99% sequence identity to a sequence or a portionof a sequence selected from the group consisting of SEQ ID NOs: 1 to 89,and further comprising a heterologous promoter and a transcriptionterminator.

In aspects according to the present disclosure, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 100 percent sequence identity to a region of 10 to 17 or morecontiguous nucleotides in the target gene or RNA transcribed from thetarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 100 percentsequence identity to a region of 18 to 25, or more contiguousnucleotides in the target gene or RNA transcribed from the target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 100 percent sequence identityto a region of 20 to 30, or more contiguous nucleotides in the targetgene or RNA transcribed from the target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionof 25 to 35, or more contiguous nucleotides in the target gene or RNAtranscribed from the target gene. In an aspect, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 100 percent sequence identity to a region of 30 to 40, or morecontiguous nucleotides in the target gene or RNA transcribed from thetarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 100 percentsequence identity to a region of 40 to 50, or more contiguousnucleotides in the target gene or RNA transcribed from the target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 100 percent sequence identityto a region of 50 to 60, or more contiguous nucleotides in the targetgene or RNA transcribed from the target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionof 45 to 60, or more contiguous nucleotides in the target gene or RNAtranscribed from the target gene. In an aspect, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 100 percent sequence identity to a region up to 60 contiguousnucleotides in the target gene or RNA transcribed from the target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 100 percent sequence identityto a region up to 50 contiguous nucleotides in the target gene or RNAtranscribed from the target gene. In an aspect, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 100 percent sequence identity to a region up to 40 contiguousnucleotides in the target gene or RNA transcribed from the target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 100 percent sequence identityto a region of at least 25 contiguous nucleotides in the target gene orRNA transcribed from the target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 100 percent sequence identity to a region of at least 35contiguous nucleotides in the target gene or RNA transcribed from thetarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 100 percentsequence identity to a region of at least 40 contiguous nucleotides inthe target gene or RNA transcribed from the target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionof at least 50 contiguous nucleotides in the target gene or RNAtranscribed from the target gene. In an aspect, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 100 percent sequence identity to a region of at least 60contiguous nucleotides in the target gene or RNA transcribed from thetarget gene. In an aspect, a target gene may be a gene comprising SEQ IDNO: 1 to 89. In an aspect, a target gene may be a gene comprising SEQ IDNO: 1. In an aspect, a target gene may be a gene comprising SEQ ID NO:2. In an aspect, a target gene may be a gene comprising SEQ ID NO: 3. Inan aspect, a target gene may be a gene comprising SEQ ID NO: 4. In anaspect, a target gene may be a gene comprising SEQ ID NO: 69. In anaspect, a target gene may be a gene comprising SEQ ID NO: 70. In anaspect, a target gene may be a gene comprising SEQ ID NO: 88. In anaspect, a target gene may be a gene comprising SEQ ID NO: 89. In anaspect, a target gene may be a gene comprising a sequence selected fromSEQ ID NOs:71-87. In an aspect, a target gene may be a gene comprising asequence selected from SEQ ID NOs:6-68.

In aspects according to the present disclosure, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 99 percent sequence identity to a region of 10 to 17 or morecontiguous nucleotides in the target gene or RNA transcribed from thetarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 99 percentsequence identity to a region of 18 to 25, or more contiguousnucleotides in the target gene or RNA transcribed from the target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 99 percent sequence identityto a region of 20 to 30, or more contiguous nucleotides in the targetgene or RNA transcribed from the target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 99 percent sequence identity to a region of25 to 35, or more contiguous nucleotides in the target gene or RNAtranscribed from the target gene. In an aspect, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 99 percent sequence identity to a region of 30 to 40, or morecontiguous nucleotides in the target gene or RNA transcribed from thetarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 99 percentsequence identity to a region of 40 to 50, or more contiguousnucleotides in the target gene or RNA transcribed from the target gene.In an aspect, an anti-parasitic, anti-pest or insecticidal compositioncomprises a nucleic acid molecule having 99 percent sequence identity toa region of 50 to 60, or more contiguous nucleotides in the target geneor RNA transcribed from the target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 99 percent sequence identity to a region of 45 to 60, ormore contiguous nucleotides in the target gene or RNA transcribed fromthe target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having99 percent sequence identity to a region up to 60 contiguous nucleotidesin the target gene or RNA transcribed from the target gene. In anaspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 99 percent sequence identityto a region up to 50 contiguous nucleotides in the target gene or RNAtranscribed from the target gene. In an aspect, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 99 percent sequence identity to a region up to 40 contiguousnucleotides in the target gene or RNA transcribed from the target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 99 percent sequence identityto a region of at least 25 contiguous nucleotides in the target gene orRNA transcribed from the target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 99 percent sequence identity to a region of at least 35contiguous nucleotides in the target gene or RNA transcribed from thetarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 99 percentsequence identity to a region of at least 40 contiguous nucleotides inthe target gene or RNA transcribed from the target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 99 percent sequence identity to a region ofat least 50 contiguous nucleotides in the target gene or RNA transcribedfrom the target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having99 percent sequence identity to a region of at least 60 contiguousnucleotides in the target gene or RNA transcribed from the target gene.In some aspects, an anti-parasitic, anti-pest or insecticidal nucleicacid has at least 98 percent sequence identity to a region of the targetgene. In an aspect, an anti-parasitic, anti-pest or insecticidal nucleicacid has at least 97 percent sequence identity to a region of the targetgene. In some aspects, an anti-parasitic, anti-pest or insecticidalnucleic acid has at least 96 percent sequence identity to a region ofthe target gene. In an aspect, an anti-parasitic, anti-pest orinsecticidal nucleic acid has at least 95 percent sequence identity to aregion of the target gene. In some aspects, an anti-parasitic, anti-pestor insecticidal nucleic acid has at least 94 percent sequence identityto a region of the target gene. In an aspect, an anti-parasitic,anti-pest or insecticidal nucleic acid has at least 93 percent sequenceidentity to a region of the target gene. In some aspects, ananti-parasitic, anti-pest or insecticidal nucleic acid has at least 92percent sequence identity to a region of the target gene. In an aspect,an anti-parasitic, anti-pest or insecticidal nucleic acid has at least91 percent sequence identity to a region of the target gene. In anaspect, an anti-parasitic, anti-pest or insecticidal nucleic acid has atleast about 83, 84, 85, 86, 87, 88, 89, 90 percent identity to a regionof the target gene as provided above. In an aspect, a target gene may bea gene comprising SEQ ID NO: 1 to 89. In an aspect, a target gene may bea gene comprising SEQ ID NO: 1. In an aspect, a target gene may be agene comprising SEQ ID NO: 2. In an aspect, a target gene may be a genecomprising SEQ ID NO: 3. In an aspect, a target gene may be a genecomprising SEQ ID NO: 4. In an aspect, a target gene may be a genecomprising SEQ ID NO: 69. In an aspect, a target gene may be a genecomprising SEQ ID NO: 70. In an aspect, a target gene may be a genecomprising SEQ ID NO: 88. In an aspect, a target gene may be a genecomprising SEQ ID NO: 89. In an aspect, a target gene may be a genecomprising sequence selected from SEQ ID NOs:71-87. In an aspect, atarget gene may be a gene comprising a sequence selected from SEQ IDNOs:6-68.

In aspects according to the present disclosure, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 100 percent sequence identity to a region of 10 to 17 or morecontiguous nucleotides in to one allele or one family member of a giventarget gene (coding or non-coding sequence of a gene). In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionof 18 to 25, or more contiguous nucleotides to one allele or one familymember of a given target gene). In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having100 percent sequence identity to a region of 20 to 30, or morecontiguous nucleotides to one allele or one family member of a giventarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 100 percentsequence identity to a region of 25 to 35, or more contiguousnucleotides to one allele or one family member of a given target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 100 percent sequence identityto a region of 30 to 40, or more contiguous nucleotides to one allele orone family member of a given target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 100 percent sequence identity to a region of 40 to 50,or more contiguous nucleotides to one allele or one family member of agiven target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having100 percent sequence identity to a region of 50 to 60, or morecontiguous nucleotides to one allele or one family member of a giventarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 100 percentsequence identity to a region of 45 to 60, or more contiguousnucleotides to one allele or one family member of a given target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 100 percent sequence identityto a region up to 60 contiguous nucleotides to one allele or one familymember of a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having100 percent sequence identity to a region up to 50 contiguousnucleotides to one allele or one family member of a given target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 100 percent sequence identityto a region up to 40 contiguous nucleotides to one allele or one familymember of a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having100 percent sequence identity to a region of at least 25 contiguousnucleotides to one allele or one family member of a given target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 100 percent sequence identityto a region of at least 35 contiguous nucleotides to one allele or onefamily member of a given target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 100 percent sequence identity to a region of at least 40contiguous nucleotides to one allele or one family member of a giventarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 100 percentsequence identity to a region of at least 50 contiguous nucleotides toone allele or one family member of a given target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionof at least 60 contiguous nucleotides to one allele or one family memberof a given target gene. In an aspect, a target gene may be a genecomprising SEQ ID NO: 1 to 89. In an aspect, a target gene may be a genecomprising SEQ ID NO: 1. In an aspect, a target gene may be a genecomprising SEQ ID NO: 2. In an aspect, a target gene may be a genecomprising SEQ ID NO: 3. In an aspect, a target gene may be a genecomprising SEQ ID NO: 4. In an aspect, a target gene may be a genecomprising SEQ ID NO: 69. In an aspect, a target gene may be a genecomprising SEQ ID NO: 70. In an aspect, a target gene may be a genecomprising SEQ ID NO: 88. In an aspect, a target gene may be a genecomprising SEQ ID NO: 89. In an aspect, a target gene may be a genecomprising a sequence selected from SEQ ID NOs:71-87. In an aspect, atarget gene may be a gene comprising a sequence selected from SEQ IDNOs:6-68.

In aspects according to the present disclosure, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 99 percent sequence identity to a region of 10 to 17 or morecontiguous nucleotides to one allele or one family member of a giventarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 99 percentsequence identity to a region of 18 to 25, or more contiguousnucleotides to one allele or one family member of a given target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 99 percent sequence identityto a region of 20 to 30, or more contiguous nucleotides to one allele orone family member of a given target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 99 percent sequence identity to a region of 25 to 35, ormore contiguous nucleotides to one allele or one family member of agiven target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having99 percent sequence identity to a region of 30 to 40, or more contiguousnucleotides to one allele or one family member of a given target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 99 percent sequence identityto a region of 40 to 50, or more contiguous nucleotides to one allele orone family member of a given target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 99 percent sequence identity to a region of 50 to 60, ormore contiguous nucleotides to one allele or one family member of agiven target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having99 percent sequence identity to a region of 45 to 60, or more contiguousnucleotides to one allele or one family member of a given target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 99 percent sequence identityto a region up to 60 contiguous nucleotides to one allele or one familymember of a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having99 percent sequence identity to a region up to 50 contiguous nucleotidesto one allele or one family member of a given target gene. In an aspect,a composition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 99 percent sequence identity to a region upto 40 contiguous nucleotides to one allele or one family member of agiven target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having99 percent sequence identity to a region of at least 25 contiguousnucleotides to one allele or one family member of a given target gene.In an aspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 99 percent sequence identityto a region of at least 35 contiguous nucleotides to one allele or onefamily member of a given target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 99 percent sequence identity to a region of at least 40contiguous nucleotides to one allele or one family member of a giventarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 99 percentsequence identity to a region of at least 50 contiguous nucleotides toone allele or one family member of a given target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 99 percent sequence identity to a region ofat least 60 contiguous nucleotides to one allele or one family member ofa given target gene. In some aspects, an anti-parasitic, anti-pest orinsecticidal nucleic acid has at least 98 percent sequence identity to aregion of the target gene. In an aspect, an anti-parasitic, anti-pest orinsecticidal nucleic acid has at least 97 percent sequence identity to aregion of the target gene. In some aspects, an anti-parasitic, anti-pestor insecticidal nucleic acid has at least 96 percent sequence identityto a region of the target gene. In an aspect, an anti-parasitic,anti-pest or insecticidal nucleic acid has at least 95 percent sequenceidentity to a region of the target gene. In some aspects, ananti-parasitic, anti-pest or insecticidal nucleic acid has at least 94percent sequence identity to a region of the target gene. In an aspect,an anti-parasitic, anti-pest or insecticidal nucleic acid has at least93 percent sequence identity to a region of the target gene. In someaspects, an anti-parasitic, anti-pest or insecticidal nucleic acid hasat least 92 percent sequence identity to a region of the target gene. Inan aspect, an anti-parasitic, anti-pest or insecticidal nucleic acid hasat least 91 percent sequence identity to a region of the target gene. Inan aspect, an anti-parasitic, anti-pest or insecticidal nucleic acid hasat least about 83, 84, 85, 86, 87, 88, 89, 90 percent identity to aregion of the target gene as provided above. In an aspect, a target genemay be a gene comprising SEQ ID NO: 1 to 89. In an aspect, a target genemay be a gene comprising SEQ ID NO: 1. In an aspect, a target gene maybe a gene comprising SEQ ID NO: 2. In an aspect, a target gene may be agene comprising SEQ ID NO: 3. In an aspect, a target gene may be a genecomprising SEQ ID NO: 4. In an aspect, a target gene may be a genecomprising SEQ ID NO: 69. In an aspect, a target gene may be a genecomprising SEQ ID NO: 70. In an aspect, a target gene may be a genecomprising SEQ ID NO: 88. In an aspect, a target gene may be a genecomprising SEQ ID NO: 89. In an aspect, a target gene may be a genecomprising a sequence selected from SEQ ID NOs:71-87. In an aspect, atarget gene may be a gene comprising a sequence selected from SEQ IDNOs:6-68.

In aspects according to the present disclosure, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 100 percent sequence identity to a region of 10 to 17 or morecontiguous nucleotides of identity with or complementarity to multiplealleles or family members of a given target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionof 18 to 25, or more contiguous nucleotides to one allele or one familymember of a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having100 percent sequence identity to a region of 20 to 30, or morecontiguous nucleotides of identity with or complementarity to multiplealleles or family members of a given target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionof 25 to 35, or more contiguous nucleotides of identity with orcomplementarity to multiple alleles or family members of a given targetgene. In an aspect, a composition comprises an anti-parasitic, anti-pestor insecticidal nucleic acid molecule having 100 percent sequenceidentity to a region of 30 to 40, or more contiguous nucleotides ofidentity with or complementarity to multiple alleles or family membersof a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having100 percent sequence identity to a region of 40 to 50, or morecontiguous nucleotides of identity with or complementarity to multiplealleles or family members of a given target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionof 50 to 60, or more contiguous nucleotides of identity with orcomplementarity to multiple alleles or family members of a given targetgene. In an aspect, a composition comprises an anti-parasitic, anti-pestor insecticidal nucleic acid molecule having 100 percent sequenceidentity to a region of 45 to 60, or more contiguous nucleotides ofidentity with or complementarity to multiple alleles or family membersof a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having100 percent sequence identity to a region up to 60 contiguousnucleotides of identity with or complementarity to multiple alleles orfamily members of a given target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 100 percent sequence identity to a region up to 50contiguous nucleotides of identity with or complementarity to multiplealleles or family members of a given target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionup to 40 contiguous nucleotides of identity with or complementarity tomultiple alleles or family members of a given target gene. In an aspect,a composition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 100 percent sequence identity to a regionof at least 25 contiguous nucleotides of identity with orcomplementarity to multiple alleles or family members of a given targetgene. In an aspect, a composition comprises an anti-parasitic, anti-pestor insecticidal nucleic acid molecule having 100 percent sequenceidentity to a region of at least 35 contiguous nucleotides of identitywith or complementarity to multiple alleles or family members of a giventarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 100 percentsequence identity to a region of at least 40 contiguous nucleotides ofidentity with or complementarity to multiple alleles or family membersof a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having100 percent sequence identity to a region of at least 50 contiguousnucleotides of identity with or complementarity to multiple alleles orfamily members of a given target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 100 percent sequence identity to a region of at least 60contiguous nucleotides of identity with or complementarity to multiplealleles or family members of a given target gene. In an aspect, a targetgene may be a gene comprising SEQ ID NO: 1 to 89. In an aspect, a targetgene may be a gene comprising SEQ ID NO: 1. In an aspect, a target genemay be a gene comprising SEQ ID NO: 2. In an aspect, a target gene maybe a gene comprising SEQ ID NO: 3. In an aspect, a target gene may be agene comprising SEQ ID NO: 4. In an aspect, a target gene may be a genecomprising SEQ ID NO: 69. In an aspect, a target gene may be a genecomprising SEQ ID NO: 70. In an aspect, a target gene may be a genecomprising SEQ ID NO: 88. In an aspect, a target gene may be a genecomprising SEQ ID NO: 89. In an aspect, a target gene may be a genecomprising a sequence selected from SEQ ID NOs:71-87. In an aspect, atarget gene may be a gene comprising a sequence selected from SEQ IDNOs:6-68.

In aspects according to the present disclosure, a composition comprisesan anti-parasitic, anti-pest or insecticidal nucleic acid moleculehaving 99 percent sequence identity to a region of 10 to 17 or morecontiguous nucleotides of identity with or complementarity to multiplealleles or family members of a given target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 99 percent sequence identity to a region of18 to 25, or more contiguous nucleotides of identity with orcomplementarity to multiple alleles or family members of a given targetgene. In an aspect, a composition comprises an anti-parasitic, anti-pestor insecticidal nucleic acid molecule having 99 percent sequenceidentity to a region of 20 to 30, or more contiguous nucleotides ofidentity with or complementarity to multiple alleles or family membersof a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having99 percent sequence identity to a region of 25 to 35, or more contiguousnucleotides of identity with or complementarity to multiple alleles orfamily members of a given target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 99 percent sequence identity to a region of 30 to 40, ormore contiguous nucleotides of identity with or complementarity tomultiple alleles or family members of a given target gene. In an aspect,a composition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 99 percent sequence identity to a region of40 to 50, or more contiguous nucleotides of identity with orcomplementarity to multiple alleles or family members of a given targetgene. In an aspect, a composition comprises an anti-parasitic, anti-pestor insecticidal nucleic acid molecule having 99 percent sequenceidentity to a region of 50 to 60, or more contiguous nucleotides ofidentity with or complementarity to multiple alleles or family membersof a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having99 percent sequence identity to a region of 45 to 60, or more contiguousnucleotides of identity with or complementarity to multiple alleles orfamily members of a given target gene. In an aspect, a compositioncomprises an anti-parasitic, anti-pest or insecticidal nucleic acidmolecule having 99 percent sequence identity to a region up to 60contiguous nucleotides of identity with or complementarity to multiplealleles or family members of a given target gene. In an aspect, acomposition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 99 percent sequence identity to a region upto 50 contiguous nucleotides of identity with or complementarity tomultiple alleles or family members of a given target gene. In an aspect,a composition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 99 percent sequence identity to a region upto 40 contiguous nucleotides of identity with or complementarity tomultiple alleles or family members of a given target gene. In an aspect,a composition comprises an anti-parasitic, anti-pest or insecticidalnucleic acid molecule having 99 percent sequence identity to a region ofat least 25 contiguous nucleotides of identity with or complementarityto multiple alleles or family members of a given target gene. In anaspect, a composition comprises an anti-parasitic, anti-pest orinsecticidal nucleic acid molecule having 99 percent sequence identityto a region of at least 35 contiguous nucleotides of identity with orcomplementarity to multiple alleles or family members of a given targetgene. In an aspect, a composition comprises an anti-parasitic, anti-pestor insecticidal nucleic acid molecule having 99 percent sequenceidentity to a region of at least 40 contiguous nucleotides of identitywith or complementarity to multiple alleles or family members of a giventarget gene. In an aspect, a composition comprises an anti-parasitic,anti-pest or insecticidal nucleic acid molecule having 99 percentsequence identity to a region of at least 50 contiguous nucleotides ofidentity with or complementarity to multiple alleles or family membersof a given target gene. In an aspect, a composition comprises ananti-parasitic, anti-pest or insecticidal nucleic acid molecule having99 percent sequence identity to a region of at least 60 contiguousnucleotides of identity with or complementarity to multiple alleles orfamily members of a given target gene. In some aspects, ananti-parasitic, anti-pest or insecticidal nucleic acid has at least 98percent sequence identity to a region of a target gene. In an aspect, ananti-parasitic, anti-pest or insecticidal nucleic acid has at least 97percent sequence identity to a region of a target gene. In some aspects,an anti-parasitic, anti-pest or insecticidal nucleic acid has at least96 percent sequence identity to a region of a target gene. In an aspect,an anti-parasitic, anti-pest or insecticidal nucleic acid has at least95 percent sequence identity to a region of a target gene. In someaspects, an anti-parasitic, anti-pest or insecticidal nucleic acid hasat least 94 percent sequence identity to a region of a target gene. Inan aspect, an anti-parasitic, anti-pest or insecticidal nucleic acid hasat least 93 percent sequence identity to a region of a target gene. Insome aspects, an anti-parasitic, anti-pest or insecticidal nucleic acidhas at least 92 percent sequence identity to a region of a target gene.In an aspect, an anti-parasitic, anti-pest or insecticidal nucleic acidhas at least 91 percent sequence identity to a region of a target gene.In an aspect, an anti-parasitic, anti-pest or insecticidal nucleic acidhas at least about 83, 84, 85, 86, 87, 88, 89, 90 percent identity to aregion of a target gene as provided above. In an aspect, a target genemay be a gene comprising SEQ ID NO: 1 to 89. In an aspect, a target genemay be a gene comprising SEQ ID NO: 1. In an aspect, a target gene maybe a gene comprising SEQ ID NO: 2. In an aspect, a target gene may be agene comprising SEQ ID NO: 3. In an aspect, a target gene may be a genecomprising SEQ ID NO: 4. In an aspect, a target gene may be a genecomprising SEQ ID NO: 69. In an aspect, a target gene may be a genecomprising SEQ ID NO: 70. In an aspect, a target gene may be a genecomprising SEQ ID NO: 88. In an aspect, a target gene may be a genecomprising SEQ ID NO: 89. In an aspect, a target gene may be a genecomprising a sequence selected from SEQ ID NOs:71-87. In an aspect, atarget gene may be a gene comprising a sequence selected from SEQ IDNOs: 6-68.

This application provides and discloses compositions comprising ananti-parasitic, anti-pest or insecticidal nucleic acid molecule and anexcipient substance. In an aspect, the excipient can be a combination ofone or more inactive components. In some aspects, the excipientcomprises a sugar. Exemplary sugars include hexoses, disaccharides,trisaccharides and higher sugars. Excipient sugars include, for example,fructose, glucose, sucrose, trehalose, lactose, galactose, ribose. Inother aspects the excipient comprises a sugar and a solvent. In otheraspects, the excipient comprises a protein. In an aspect, the protein isa soy protein. In other aspects the excipient may be pollen. In aspectsaccording to the present disclosure, the excipient may be a bee food. Insome aspects, the excipient comprises Tryptone. In some aspects, theexcipient comprises yeast extract. In some aspects, the excipientcomprises an essential oil.

Bee feeding is common practice amongst bee-keepers, for providing bothnutritional and other, for example, supplemental needs. Bees typicallyfeed on honey and pollen, but have been known to ingest non-naturalfeeds as well. Bees can be fed various foodstuffs including, but notlimited to Wheast (a dairy yeast grown on cottage cheese), soybeanflour, yeast (e.g. brewer's yeast, torula yeast) and yeast productsproducts-fed singly or in combination and soybean flour fed as a dry mixor moist cake inside the hive or as a dry mix in open feeders outsidethe hive. Also useful is sugar, or a sugar syrup. The addition of 10 to12 percent pollen to a supplement fed to bees improves palatability. Theaddition of 25 to percent pollen improves the quality and quantity ofessential nutrients that are required by bees for vital activity. Caneor beet sugar, isomerized corn syrup, and type-50 sugar syrup aresatisfactory substitutes for honey in the natural diet of honey bees.The last two can be supplied only as a liquid to bees. Liquid feed canbe supplied to bees inside the hive by, for example, any of thefollowing methods: friction-top pail, combs within the brood chamber,division board feeder, boardman feeder, etc. Dry sugar may be fed byplacing a pound or two on the inverted inner cover. A supply of watermust be available to bees at all times. In one aspect, pan or trays inwhich floating supports-such as wood chips, cork, or plastic sponge-arepresent are envisaged. Detailed descriptions of supplemental feeds forbees can be found in, for example, USDA publication by Standifer, et al.1977, entitled “Supplemental Feeding of Honey Bee Colonies” (USDA,Agriculture Information Bulletin No. 413).

In aspects according to the present disclosure, an anti-parasitic,anti-pest or insecticidal nucleic acid, for example a dsRNA, isabsorbable. As used herein “absorbable,” refers to mechanisms theprovide for the uptake of a nucleic acid that is not by ingestion. In anaspect, an anti-parasitic, anti-pest or insecticidal nucleic acid may beabsorbed through the skin of an organism, or the exoskeleton of anarthropod. In an aspect, an absorbable nucleic acid is dissolved in anexcipient. In other aspects, an absorbable nucleic acid is suspended inan excipient. Excipients for solvation or suspension may be aqueous ornon-aqueous. In some aspects, the anti-parasitic, anti-pest orinsecticidal nucleic acid is absorbed by a host organism and transferredto a parasitic organism by feeding. In other aspects, theanti-parasitic, anti-pest or insecticidal nucleic acid is absorbed by ahost organism and transferred to a parasitic organism by absorption. Inan aspect, an anti-parasitic, anti-pest or insecticidal nucleic acid ofthe present disclosure is absorbed directly by the parasite.

In aspects according to the present disclosure an anti-parasitic,anti-pest or insecticidal nucleic acid, for example a dsRNA, is combinedwith an excipient. In an aspect, the nucleic acid may be provided as aratio of nucleic acid to excipient. In an aspect, the ratio may be onepart nucleic acid to 4 parts excipient. In an aspect the ratio ofnucleic acid to excipient may be 1:1, 1:2, 1:5, or 1:10. In otheraspects, the ratio of nucleic acid to excipient may be 1:20, 1:25, 1:30,1:40, or more. In an aspect, ratio of nucleic acid to excipient may be1:50. In aspects according to the present disclosure, the ratio may bedetermined as a volume to volume (v/v) ratio, a weight:weight (w/w)ratio. In certain aspects, the ratio may be expressed as a weight:volume(w/v) ratio. In certain aspects, a nucleic acid and an excipient may bea dsRNA and an excipient.

In aspects according to the present disclosure, the composition maycomprise a weight of an anti-parasitic, anti-pest or insecticidalnucleic acid combined with an excipient. In an aspect, the nucleic acidmay comprise a percentage of the total weight of the composition. In anaspect, the nucleic acid may comprise about 0.1% by weight of thecomposition. In an aspect, the nucleic acid may comprise about 0.2% byweight of the composition. In an aspect, the nucleic acid may compriseabout 0.3% by weight of the composition. In another aspect, the nucleicacid may comprise about 0.4% by weight of the composition. In an aspect,the nucleic acid may comprise up to 0.5% by weight of the composition.In an aspect, the nucleic acid may comprise up to 0.6% by weight of thecomposition. In an aspect, the nucleic acid may comprise up to 0.7% byweight of the composition. In an aspect, the nucleic acid may compriseup to 0.8% by weight of the composition. In another aspect, the nucleicacid may comprise up to 1.0% by weight of the composition. In otheraspects, the nucleic acid may comprise up to 1.5% by weight of thecomposition. In yet other aspects, the nucleic acid may comprise up to2.0% by weight, or 2.5% by weight of the composition. In certainaspects, a nucleic acid and an excipient may be a dsRNA and anexcipient.

The present disclosure provides for, and includes, compositions havingfrom 0.1% to 5% by weight of one or more anti-parasitic, anti-pest orinsecticidal nucleic acids. In other aspects, a composition may comprisefrom 0.1 to 4%, 0.1 to 3%, 0.1 to 2%, 0.1 to 1%, 0.1 to 2%, 0.1 to 3%,or 0.1 to 4% by weight nucleic acid. In an aspect, a composition maycomprise from 0.2% to 5% by weight nucleic acid. In other aspects, acomposition may comprise from 0.2 to 4%, 0.2 to 3%, 0.2 to 2%, 0.2 to1%, 0.2 to 2%, 0.2 to 3%, or 0.2 to 4% by weight nucleic acid. In otheraspects, a composition may comprise up to 1%, up to 2%, up to 3%, up to4%, or up to 5% nucleic acid. In other aspects, a composition maycomprise up to 7.5%, up to 10%, or up to 15% nucleic acid. In certainaspects, a nucleic acid and an excipient may be a dsRNA and anexcipient.

The present disclosure provides for, and includes, compositions havingfrom 0.1 to 10 mg/ml of one ore more anti-parasitic, anti-pest orinsecticidal nucleic acids. In other aspects, a composition may comprisefrom 0.1 to 1.0 mg/ml, 0.1 to 2.0 mg/ml, 0.1 to 2.5 mg/ml, 0.1 to 5mg/ml, 0.1 to 10 mg/ml, 0.1 to 15 mg/ml, or 0.1 to 20 mg/ml nucleicacid. In certain aspects, a composition may comprise at least 0.1 μg/mlnucleic acid. In certain other aspects, a composition may comprise atleast 1.0 μg/ml nucleic acid. In yet other aspects, a composition maycomprise at least 10 μg/ml nucleic acid. In an aspect, a composition maycomprise from 0.5 to 10 mg/ml nucleic acid. In other aspects, acomposition may comprise from 0.5 to 1.0 mg/ml, 0.5 to 2.0 mg/ml, 0.5 to2.5 mg/ml, 0.5 to 5 mg/ml, 0.5 to 10 mg/ml, 0.5 to 15 mg/ml, or 0.5 to20 mg/ml nucleic acid. In an aspect, a composition may comprise from 1.0to 10 mg/ml nucleic acid. In other aspects, a composition may comprisefrom 1.0 to 2.0 mg/ml, 1.0 to 2.5 mg/ml, 1.0 to 5 mg/ml, 1.0 to 10mg/ml, 1.0 to 15 mg/ml, or 1.0 to 20 mg/ml nucleic acid. In certainaspects, the anti-parasitic, anti-pest or insecticidal nucleic acid inthe composition comprises a dsRNA.

The present disclosure, provides for, and includes selective insecticidecompositions and methods of using selective insecticide compositions.

As used herein, a “selective insecticide composition,” is a compositionthat is more effective for one or more arthropod species and is lesseffective for one or more different arthropod species. A selectiveinsecticide composition includes compositions that kill adults orimmature arthropods and includes compositions that are larvicides andovicides. A selective insecticide may be a systemic insecticidesincorporated by treated food, including the blood or hemolymph obtainedfrom a host organisms. A selective insecticide may be a contactinsecticides are toxic to certain insects brought into direct contact,and are non-toxic or minimally toxic to certain other insects. In someembodiments, a selective insecticide composition is anti-pest. In someembodiments, a selective insecticide composition is anti-parasitic. Insome embodiments, a selective insecticide composition is a miticide. Insome embodiments, a selective insecticide composition is toxic to atargeted parasitic or pest insect and non-toxic or minimally toxic tonon-target organisms. Examples of non-target organisms include, but arenot limited to beneficial insects, nematodes, birds, mammals, andplants. In some embodiments, a selective insecticide composition istoxic to a parasitic insect, for example Varroa mite, and non-toxic orminimally toxic to the host organism, for example bees. In someembodiments, a selective insecticide composition is toxic to one or morepest or parasitic insects selected from the group consisting of: Varroadestructor, Ixodes scapularis, Solenopsis invicta, Tetranychus urticae,Aedes aegypti, Culex quinquefasciatus, Acyrthosiphon pisum, andPediculus humanus.

In certain aspects according to the present disclosure, a selectiveinsecticide may be incorporated into a bacteria or yeast by geneticmodification (for example, a transgenic bacteria or yeast engineered toexpress a nucleic acid of the present disclosure). A selectiveinsecticide introduced by genetic modification of a bacteria or yeastmay act directly on the pest organism, or indirectly by being ingestedby a host of the pest organism.

In an aspect according to the present disclosure, a selectiveinsecticide may be a more effective insecticide against one or morefirst insects than against one or more second insects. In an aspect, aselective insecticide may be toxic to a first insect and have no effecton a second insect. In an aspect, a selective insecticide may be toxicto a first insect and require significantly higher concentrations oramounts to have an effect on a second insect. In an aspect, a selectiveinsecticide may be 2 times or more toxic to a first insect compared to asecond insect. In an aspect, a selective insecticide may be 4 times ormore toxic to a first insect compared to a second insect. In an aspect,a selective insecticide may be 5 times or more toxic to a first insectcompared to a second insect. In an aspect, a selective insecticide maybe 10 times or more toxic to a first insect compared to a second insect.

In an aspect, a selective insecticide may inhibit the growth,development or fecundity of a first insect and have no effect on asecond insect. In an aspect, a selective insecticide may inhibit thegrowth, development or fecundity a first insect and requiresignificantly higher concentrations or amounts to have a similar effecton a second insect. In an aspect, a selective insecticide may require 2times or more of the active ingredient to inhibit the growth,development or fecundity of a second insect. In an aspect, a selectiveinsecticide may require 4 times or more of the active ingredient toinhibit the growth, development or fecundity of a second insect. In anaspect, a selective insecticide may require 5 times or more of theactive ingredient to inhibit the growth, development or fecundity of asecond insect. In an aspect, a selective insecticide may require 10times or more of the active ingredient to inhibit the growth,development or fecundity of a second insect.

The present disclosure further includes, and provides for, methods oftreating or preventing Colony Collapse Disorder in a honeybee colony,comprising providing an effective amount of a composition comprising anucleic acid that is essentially identical or essentially complementaryto a region of a Varroa destructor calmodulin gene sequence to ahoneybee whereby the level of Varroa destructor infestation is reduced.In an aspect, the method comprises providing an effective amount of acomposition comprising a nucleic acid that is essentially identical oressentially complementary to at least 19 contiguous nucleotides of SEQID NO: 1. In an aspect, the method comprises providing an effectiveamount of a composition comprising a nucleic acid that is essentiallyidentical or essentially complementary to at least 19 contiguousnucleotides of SEQ ID NO: 2. In an aspect, the method comprisesproviding an effective amount of a composition comprising a nucleic acidthat is essentially identical or essentially complementary to at least19 contiguous nucleotides of SEQ ID NO: 69. In an aspect, the methodcomprises providing an effective amount of a composition comprising anucleic acid that is essentially identical or essentially complementaryto at least 19 contiguous nucleotides of SEQ ID NO: 70. In an aspect,the method comprises providing an effective amount of a compositioncomprising a nucleic acid according to SEQ ID NO: 3. In an aspect, themethod comprises providing an effective amount of a compositioncomprising a nucleic acid according to SEQ ID NO: 4. In an aspect, themethod comprises providing an effective amount of a compositioncomprising a nucleic acid according to SEQ ID NO: 88. In an aspect, themethod comprises providing an effective amount of a compositioncomprising a nucleic acid according to SEQ ID NO: 89. In an aspect, themethod comprises providing an effective amount of a compositioncomprising two or more nucleic acids having a sequence selected from thegroup consisting of: SEQ ID NOs: 3, 4, 88 and 89. In an aspect, themethod comprises providing an effective amount of a compositioncomprising a nucleic acid that is essentially identical or essentiallycomplementary to at least 19 contiguous nucleotides of a sequenceselected from SEQ ID NOs: 71-87. In an aspect, the method comprisesproviding an effective amount of a composition comprising a nucleic acidthat is essentially identical or essentially complementary to at least23 contiguous nucleotides of a sequence selected from SEQ ID NOs: 71-87.In an aspect, the method comprises providing an effective amount of acomposition comprising a nucleic acid that is essentially identical oressentially complementary to at least 30 contiguous nucleotides of asequence selected from SEQ ID NOs: 71-87. In an aspect, the methodcomprises providing an effective amount of a composition comprising anucleic acid that is essentially identical or essentially complementaryto at least 40 contiguous nucleotides of a sequence selected from SEQ IDNOs: 71-87. In an aspect, the method comprises providing an effectiveamount of a composition comprising a nucleic acid that is essentiallyidentical or essentially complementary to at least 50 contiguousnucleotides of a sequence selected from SEQ ID NOs: 71-87. In an aspect,the method comprises providing an effective amount of a compositioncomprising a nucleic acid that is essentially identical or essentiallycomplementary to at least 60 contiguous nucleotides of a sequenceselected from SEQ ID NOs: 71-87. In an aspect, the method comprisesproviding an effective amount of a composition comprising a nucleic acidthat is essentially identical or essentially complementary to at least70 contiguous nucleotides of a sequence selected from SEQ ID NOs: 71-87.In an aspect, the method comprises providing an effective amount of acomposition comprising a nucleic acid that is essentially identical oressentially complementary to at least 80 contiguous nucleotides of asequence selected from SEQ ID NOs: 71-87. In an aspect, the methodcomprises providing an effective amount of a composition comprising anucleic acid that is essentially identical or essentially complementaryto at least 90 contiguous nucleotides of a sequence selected from SEQ IDNOs: 71-87. In an aspect, the method comprises providing an effectiveamount of a composition comprising a nucleic acid that is essentiallyidentical or essentially complementary to at least 100 contiguousnucleotides of a sequence selected from SEQ ID NOs: 71-87. In an aspect,the method comprises providing an effective amount of a compositioncomprising a nucleic acid that is essentially identical or essentiallycomplementary to at least 110 contiguous nucleotides of a sequenceselected from SEQ ID NOs: 71-87. In an aspect, the method comprisesproviding an effective amount of a composition comprising a nucleic acidthat is essentially identical or essentially complementary to at least120 contiguous nucleotides of a sequence selected from SEQ ID NOs:71-87. In an aspect, the method comprises providing an effective amountof a composition comprising a nucleic acid that is essentially identicalor essentially complementary to at least 130 contiguous nucleotides of asequence selected from SEQ ID NOs: 71-87. In an aspect, the methodcomprises providing an effective amount of a composition comprising anucleic acid that is essentially identical or essentially complementaryto at least 140 contiguous nucleotides of a sequence selected from SEQID NOs: 71-87. In an aspect, the method comprises providing an effectiveamount of a composition comprising a nucleic acid that is essentiallyidentical or essentially complementary to a sequence selected from SEQID NOs: 71-87. In an aspect, the method comprises providing an effectiveamount of a composition comprising a nucleic acid according to asequence selected from SEQ ID NOs: 71-87.

The present disclosure provides for, and includes, methods for reducingthe parasite load of a host organism. In an aspect, the parasite loadrefers to the number of parasites per individual host. In an aspect, theparasite load refers to the average number of parasites per 100 hostorganisms. In an aspect, the parasite load may refer to the number ofparasites per colony of parasite hosts. In aspects according to thepresent disclosure the parasite is Varroa destructor and the host is thehoney bee, Apis mellifera. In certain aspects, the parasite load refersto the number of Varroa destructor parasites per 100 honeybees in acolony. In some embodiments, the present disclosure provides for, andincludes, methods and compositions for reducing the parasite load toless than 6 Varroa destructor parasites per 100 honeybees in a colony.In some embodiments, the present disclosure provides for, and includes,methods and compositions for reducing the parasite load to less than 5Varroa destructor parasites per 100 honeybees in a colony. In someembodiments, the present disclosure provides for, and includes, methodsand compositions for reducing the parasite load to less than 4 Varroadestructor parasites per 100 honeybees in a colony. In some embodiments,the present disclosure provides for, and includes, methods andcompositions for reducing the parasite load to less than 2 Varroadestructor parasites per 100 honeybees in a colony.

In an aspect, the methods of reducing a parasite load comprisesproviding an effective amount of an anti-parasitic, anti-pest orinsecticidal nucleic acid composition to a host organism. An effectiveamount of a composition of the present disclosure results in a decreasein the parasite load over a period of time. In an aspect, a decrease inparasite load may measured within one day of providing an effectiveamount of a nucleic acid composition. In an aspect, the parasite loadmay be measured after two days. In an aspect, the parasite load may bemeasured after 3 days. In other aspects, the parasite load may bemeasured after 5 days or after 1 week. In another aspect, the parasiteload may be measured more than one time, for example every 3 days, every5 days, every week or once a month. In certain aspects, according to thepresent disclosure, a decrease in the number of parasites may bemeasured and compared to an untreated control organism or colony. Inaspects according to the present disclosure the parasite is Varroadestructor and the host is the honey bee, Apis mellifera.

In aspects according to the present disclosure, a reduction in parasiteload after a period of time means a decrease in the number of parasites.In an aspect, the number of parasites may decrease by 10%, 20%, 30% ormore between measurements. In another aspect, the number of parasitesmay decrease by 40% or more between measurements. In another aspect, thenumber of parasites may decrease by 50% or more between measurements. Inanother aspect, the number of parasites may decrease by 60% or morebetween measurements. In another aspect, the number of parasites maydecrease by 70% or more between measurements. In another aspect, thenumber of parasites may decrease by 80% or more between measurements. Inanother aspect, the number of parasites may decrease by 90% or morebetween measurements.

In other aspects, the parasite load may be measured as the averagenumber of parasites per host organism. In an aspect, a decreasedparasitic load may comprise fewer than 20 parasites per 100 hostorganisms. In an aspect, a decreased parasitic load may comprise fewerthan 15 parasites per 100 host organisms. In an aspect, a decreasedparasitic load may comprise fewer than 10 parasites per 100 hostorganisms. In an aspect, a decreased parasitic load may comprise fewerthan 5 parasites per 100 host organisms. In an aspect, a decreasedparasitic load may comprise fewer than 4 parasites per 100 hostorganisms. In an aspect, a decreased parasitic load may comprise fewerthan 3 parasites per 100 host organisms. In an aspect, a decreasedparasitic load may comprise fewer than 2 parasites per 100 hostorganisms. In an aspect, a decreased parasitic load may comprise fewerthan 1 parasite per 100 host organisms. In an aspect, a decreasedparasitic load may comprise fewer than 20 parasites per 1000 hostorganisms. In an aspect, a decreased parasitic load may comprise fewerthan 15 parasites per 1000 host organisms. In an aspect, a decreasedparasitic load may comprise fewer than 10 parasites per 1000 hostorganisms. In an aspect, a decreased parasitic load may comprise fewerthan 5 parasites per 1000 host organisms. In an aspect, a decreasedparasitic load may comprise fewer than 4 parasites per 1000 hostorganisms. In an aspect, a decreased parasitic load may comprise fewerthan 3 parasites per 1000 host organisms. In an aspect, a decreasedparasitic load may comprise fewer than 2 parasites per 1000 hostorganisms. In an aspect, a decreased parasitic load may comprise fewerthan 1 parasite per 1000 host organisms.

In aspects according to the present disclosure, a colony of hostorganisms has an initial parasite load, prior to being provided a sourceof an effective amount of a nucleic acid. In an aspect, an initialparasite load may comprise fewer than 20 parasites per 100 hostorganisms. In an aspect, an initial parasite load may comprise fewerthan 15 parasites per 100 host organisms. In an aspect, an initialparasite load may comprise fewer than 10 parasites per 100 hostorganisms. In an aspect, an initial parasite load may comprise fewerthan 5 parasites per 100 host organisms.

In an aspect, an initial parasite load may comprise fewer than 4parasites per 100 host organisms. In an aspect, an initial parasite loadmay comprise fewer than 3 parasites per 100 host organisms. In anaspect, an initial parasite load may comprise fewer than 2 parasites per100 host organisms. In an aspect, an initial parasite load may comprisefewer than 1 parasite per 100 host organisms.

In aspects according to the present disclosure, an effective amount maybe provided periodically or continually. In an aspect, an effectiveamount of an anti-parasitic, anti-pest or insecticidal nucleic acidcomposition may be provided once, twice or three times a day. In otheraspects, an effective amount of an anti-parasitic, anti-pest orinsecticidal nucleic acid composition may be provided once a day. Inanother aspect, an effective amount of an anti-parasitic, anti-pest orinsecticidal nucleic acid composition may be provided one or more timesevery other day. In an aspect, an effective amount of an anti-parasitic,anti-pest or insecticidal nucleic acid composition may be provided everytwo days, every three days, or once a week. In an aspect, an effectiveamount of an anti-parasitic, anti-pest or insecticidal nucleic acidcomposition may be provided every two weeks. In an aspect, an effectiveamount of an anti-parasitic, anti-pest or insecticidal nucleic acidcomposition may be provided every three weeks. In an aspect, aneffective amount of an anti-parasitic, anti-pest or insecticidal nucleicacid composition may be provided once a month. In an aspect, aneffective amount of an anti-parasitic, anti-pest or insecticidal nucleicacid composition may be provided every two months. In an aspect, aneffective amount of a nucleic acid composition may be providedcontinuously to an organism in need, for example by providing acontinuous source of food. In one aspect, an effective amount of anucleic acid composition may be provided continuously as abee-ingestible composition. In aspects according to the presentdisclosure the parasite is Varroa destructor and the host is the honeybee, Apis mellifera. In aspects according to the present disclosure, ananti-parasitic, anti-pest or insecticidal nucleic acid may be a dsRNA.

In aspects according to the present disclosure, the parasitic load maydecrease over a period of time. In an aspect, the time period necessaryfor a parasitic load decrease may be 15 weeks. In another aspect, thetime period for a parasitic load decrease may be 12 weeks. In an aspect,the parasitic load decrease occurs of a period of 10 weeks. In anaspect, the time period necessary for a parasitic load decrease may be 5weeks. In another aspect, the time period for a parasitic load decreasemay be 2 weeks. In an aspect, the parasitic load decrease occurs of aperiod of 1 weeks. In some aspects, the parasitic load may decreaseafter one day, two days or three days.

The present disclosure provides for methods of reducing the parasitationof a honey bee colony comprising providing a bee colony an effectiveamount of an anti-parasitic, anti-pest or insecticidal nucleic acidcomposition. An effective amount of a composition of the presentdisclosure results in a reduction of parasitation over a period of time.In an aspect, a reduction of parasitation may measured within one day ofproviding an effective amount of an anti-parasitic, anti-pest orinsecticidal nucleic acid composition. In an aspect, the reduction ofparasitation may be measured after two days. In an aspect, the reductionof parasitation may be measured after 3 days. In other aspects, thereduction of parasitation may be measured after 5 days or after 1 week.In another aspect, the reduction of parasitation may be measured morethan one time, for example every 3 days, every 5 days, every week oronce a month. In certain aspects, according to the present disclosure, areduction of parasitation may be measured and compared to an untreatedcontrol organism or colony.

In aspects according to the present disclosure, a reduction ofparasitation after a period of time means a decrease in the total numberof parasites. In an aspect, the number of parasites may decrease by 10%,20%, 30% or more between measurements. In another aspect, the number ofparasites may decrease by 40% or more between measurements. In anotheraspect, the number of parasites may decrease by 50% or more betweenmeasurements. In another aspect, the number of parasites may decrease by60% or more between measurements. In another aspect, the number ofparasites may decrease by 70% or more between measurements. In anotheraspect, the number of parasites may decrease by 80% or more betweenmeasurements. In another aspect, the number of parasites may decrease by90% or more between measurements.

In other aspects, reduction of parasitation may be measured as theaverage number of parasites per host organism. In an aspect, a reductionof parasitation may comprise fewer than 20 parasites per 100 hostorganisms. In an aspect, a reduction of parasitation may comprise fewerthan 15 parasites per 100 host organisms. In an aspect, a reduction ofparasitation may comprise fewer than 10 parasites per 100 hostorganisms. In an aspect, a reduction of parasitation may comprise fewerthan 5 parasites per 100 host organisms. In an aspect, a reduction ofparasitation may comprise fewer than 4 parasites per 100 host organisms.In an aspect, a reduction of parasitation may comprise fewer than 3parasites per 100 host organisms. In an aspect, a reduction ofparasitation may comprise fewer than 2 parasites per 100 host organisms.In an aspect, a reduction of parasitation may comprise fewer than 1parasite per 100 host organisms.

In aspects according to the present disclosure, an effective amount ofan anti-parasitic, anti-pest or insecticidal nucleic acid resulting in areduction of parasitation may be provided periodically or continually.In an aspect, an effective amount of a nucleic acid composition may beprovided once, twice or three times a day. In other aspects, aneffective amount of an anti-parasitic, anti-pest or insecticidal nucleicacid composition may be provided once a day. In another aspect, aneffective amount of an anti-parasitic, anti-pest or insecticidal nucleicacid composition may be provided one or more times every other day. Inan aspect, an effective amount of an anti-parasitic, anti-pest orinsecticidal nucleic acid composition may be provided provide every twodays, every three days, or once a week. In an aspect, an effectiveamount of an anti-parasitic, anti-pest or insecticidal nucleic acidcomposition may be provided continuously to an organism in need, forexample by providing a continuous source of food. In one aspect, aneffective amount of an anti-parasitic, anti-pest or insecticidal nucleicacid composition may be provided continuously as a bee-ingestiblecomposition. In aspects according to the present disclosure the parasiteis Varroa destructor and the host is the honey bee, Apis mellifera. Inaspects according to the present disclosure, an anti-parasitic,anti-pest or insecticidal nucleic acid may be a dsRNA.

In aspects according to the present disclosure, the reduction ofparasitation may decrease over a period of time. In an aspect, the timeperiod necessary for a reduction of parasitation may be 15 weeks. Inanother aspect, the time period for a reduction of parasitation may be12 weeks. In an aspect, the reduction of parasitation occurs of a periodof 10 weeks. In an aspect, the time period necessary for a reduction ofparasitation may be 5 weeks. In another aspect, the time period for areduction of parasitation may be 2 weeks. In an aspect, the reduction ofparasitation occurs of a period of 1 weeks. In some aspects, thereduction of parasitation may occur after one day, two days or threedays.

In aspects according to the present disclosure, a reduction ofparasitation is measured by the number of surviving parasites ascompared to an initial measurement of the number of parasites in acolony of host organisms. In an aspect, the parasite may be a Varroadestructor mite and the host may be a honey bee, Apis mellifera. In anaspect, the number of surviving parasites may be 25% of the initialnumber of parasites. In an aspect, the number of surviving parasites maybe 15% of the initial number of parasites. In an aspect, the number ofsurviving parasites may be 10% of the initial number of parasites. In anaspect, the number of surviving parasites may be 5% of the initialnumber of parasites. In an aspect the number of surviving parasites maybe less than 5% or even undetectable after providing a host colony aneffective amount of an anti-parasitic, anti-pest or insecticidal nucleicacid composition.

In an aspect, the present disclosure provides for methods andcompositions for reducing the susceptibility of bees to Varroa miteinfestation. In other aspects, the present disclosure provides formethods and compositions to prevent the infestation of colonies of bees.In another aspect, the present disclosure provides methods andcompositions for reducing the parasitation of honeybees by the miteVarroa destructor.

According to the present disclosure, a host organism provided with asource of an anti-parasitic, anti-pest or insecticidal nucleic acid, canaccumulate nucleic acid in the host body, usually the hemolymph. Byharboring nucleic acid, such host organisms become resistant, or lesssusceptible to parasitation. In other aspects, a colony of hostorganisms, provided with a source of nucleic acid, can accumulatenucleic acid in the host body of multiple members of the colony, therebyproviding resistance or decreased susceptibility to a parasite. nucleicacid found in host organisms provided with a source of nucleic acid, canbe detected using methods known to those of ordinary skill in the art.In aspects according to the present disclosure, an anti-parasitic,anti-pest or insecticidal nucleic acid may be a dsRNA.

In an aspect of the present disclosure, methods and compositions fortreating Varroa mite infestations in bees by down-regulating calmodulinand calmodulin related Varroa mite gene products, are provided. In anaspect, the compositions comprise an anti-parasitic, anti-pest orinsecticidal nucleic acid corresponding to the Varroa destructorcalmodulin sequence of SEQ ID NO: 1. In an aspect, the compositionscomprise an anti-parasitic, anti-pest or insecticidal nucleic acidcorresponding to the Varroa destructor calmodulin sequence of SEQ ID NO:2. In an aspect, the compositions comprise an anti-parasitic, anti-pestor insecticidal nucleic acid corresponding to the Varroa destructorcalmodulin sequence of SEQ ID NO: 69. In an aspect, the compositionscomprise an anti-parasitic, anti-pest or insecticidal nucleic acidcorresponding to the Varroa destructor calmodulin sequence of SEQ ID NO:70. In some aspects, the compositions comprise an anti-parasitic,anti-pest or insecticidal nucleic acid corresponding to a Varroadestructor calmodulin sequence selected from SEQ ID NOs: 71-87. Inanother aspect, the compositions comprise an anti-parasitic, anti-pestor insecticidal nucleic acid corresponding to a Varroa destructorcalmodulin sequence selected from SEQ ID NOs: 3, 4, 88 and 89. In anaspect, the compositions comprise a small RNA corresponding to theVarroa destructor calmodulin sequence of SEQ ID NO: 1. In an aspect, thecompositions comprise a small RNA corresponding to the Varroa destructorcalmodulin sequence of SEQ ID NO: 2. In an aspect, the compositionscomprise a small RNA corresponding to the Varroa destructor calmodulinsequence of SEQ ID NO: 69. In an aspect, the compositions comprise asmall RNA corresponding to the Varroa destructor calmodulin sequence ofSEQ ID NO: 70. In some aspects, the compositions comprise a small RNAcorresponding to a Varroa destructor calmodulin sequence selected fromSEQ ID NOs: 71-87. In another aspect, the compositions comprise a smallRNA corresponding to a Varroa destructor calmodulin sequence selectedfrom SEQ ID NOs: 3, 4, 88 and 89. In an aspect, the compositionscomprise a dsRNA corresponding to the Varroa destructor calmodulinsequence of SEQ ID NO: 1. In an aspect, the compositions comprise adsRNA corresponding to the Varroa destructor calmodulin sequence of SEQID NO: 2. In an aspect, the compositions comprise a dsRNA correspondingto the Varroa destructor calmodulin sequence of SEQ ID NO: 69. In anaspect, the compositions comprise a dsRNA corresponding to the Varroadestructor calmodulin sequence of SEQ ID NO: 70. In some aspects, thecompositions comprise a dsRNA corresponding to a Varroa destructorcalmodulin sequence selected from SEQ ID NOs: 71-87. In another aspect,the compositions comprise a dsRNA corresponding to a Varroa destructorcalmodulin sequence selected from SEQ ID NOs: 3, 4, 88 and 89. In anaspect, the compositions comprise an siRNA corresponding to the Varroadestructor calmodulin sequence of SEQ ID NO: 1. In an aspect, thecompositions comprise a siRNA corresponding to the Varroa destructorcalmodulin sequence of SEQ ID NO: 2. In an aspect, the compositionscomprise a siRNA corresponding to the Varroa destructor calmodulinsequence of SEQ ID NO: 69. In an aspect, the compositions comprise asiRNA corresponding to the Varroa destructor calmodulin sequence of SEQID NO: 70. In some aspects, the compositions comprise a siRNAcorresponding to a Varroa destructor calmodulin sequence selected fromSEQ ID NOs: 71-87. In another aspect, the compositions comprise a siRNAcorresponding to a Varroa destructor calmodulin sequence selected fromSEQ ID NOs: 3, 4, 88 and 89. In aspects according to the presentdisclosure the composition may comprise an anti-parasitic, anti-pest orinsecticidal nucleic acid corresponding to a region of SEQ ID NO: 1 or2. In other aspects according to the present disclosure the compositionmay comprise an anti-parasitic, anti-pest or insecticidal nucleic acidcorresponding to a region of SEQ ID NO: 69 or 70. In yet other aspectsaccording to the present disclosure the composition may comprise anucleic acid corresponding to a region of a sequence selected from SEQID NOs: 3, 4, 88 and 89.

Varroa mites parasitize pupae and adult bees and reproduce in the pupalbrood cells. The mites use their mouths to puncture the exoskeleton andfeed on the bee's hemolymph. The present inventors unexpectedly foundthat polynucleotide agents administered to the bees to treat Varroa miteinfestations presented in the bee's hemolymph thereby becoming availableto the mite.

The present inventors have shown that calmodulin-targeting dsRNAfragments can successfully be transferred to Varroa mites (see, e.g.,FIG. 2), that the dsRNA can serve to down-regulate expression ofcalmodulin genes in the Varroa mite (see, e.g., FIG. 3A) and furtherthat targeting of calmodulin genes for down-regulation can result in areduction in the number of Varroa mites (see, e.g., FIG. 3B).

Thus, according to one aspect of the present disclosure there isprovided a method of preventing or treating a Varroa destructor miteinfestation of a bee, the method comprising administering to the bee aneffective amount of a nucleic acid agent comprising a nucleic acidsequence which downregulates expression of a calmodulin gene of a Varroadestructor mite, thereby preventing or treating a Varroa destructor miteinfestation of a bee.

According to this aspect of the present disclosure the agents of thepresent disclosure are used to prevent the Varroa destructor mite fromliving as a parasite on the bee, or larvae thereof. The phrase “Varroadestructor mite” refers to the external parasitic mite that attackshoney bees Apis cerana and Apis mellifera. The mite may be at an adultstage, feeding off the bee, or at a larval stage, inside the honey beebrood cell.

As mentioned, the agents of the present disclosure are capable ofselectively down-regulating expression of a gene product of a Varroadestructor mite. As used herein, the phrase “gene product” refers to anRNA molecule or a protein. According to one aspect, the Varroadestructor mite gene product is one which is essential for miteviability. Down-regulation of such a gene product would typically resultin killing of the Varroa mite. According to another aspect, the Varroadestructor mite gene product is one which is essential for mitereproduction. Down-regulation of such a gene product would typicallyresult in the prevention of reproduction of the Varroa mite and theeventual extermination of the mite population. According to yet anotheraspect, the Varroa destructor mite gene product is one which is requiredto generate pathogenic symptoms in the bee. In some aspects, the Varroadestructor gene product is a calmodulin gene. In certain aspects, thecalmodulin gene may comprise a nucleic acid sequence according to SEQ IDNO: 1 or SEQ ID NO: 2. In certain aspects, the calmodulin gene maycomprise a nucleic acid sequence according to SEQ ID NO: 69 or SEQ IDNO: 70.

Examples of gene products that may be down-regulated according to thisaspect of the present disclosure include, but are not limited to acalmodulin gene.

In an aspect according to the present disclosure, agents capable ofdown-regulating expression of a gene product of a Varroa destructor miteor other parasite, may downregulate to a lesser extent expression of thegene product in other animals, such as the bee or other non-targetorganism. Accordingly, certain agents of the present disclosure are ableto distinguish between the mite gene and the bee gene, down-regulatingthe former to a greater extent than the latter. In some aspects, certainagents of the present disclosure are able to distinguish between thetarget gene in the target organism and orthologs in non-targetorganisms, down-regulating the former to a greater extent than thelatter. In other aspects, the target gene of the parasite isdownregulated while the homologous host gene is not. In yet anotheraspect, the target gene of the parasite does not have a homologue in thehost. According to another aspect the agents of the present disclosuredo not down-regulate the bee gene whatsoever. For example, this may beeffected by targeting a gene that is expressed differentially in themite and not in the bee e.g. the mite sodium channel gene—FJ216963.Alternatively, the agents of the present disclosure may be targeted tomite-specific sequences of a gene that is expressed both in the mite andin the bee.

According to one aspect, the agents of the present disclosure targetsegments of Varroa genes that are at least 100 bases long and do notcarry any sequence longer than 19 bases that is entirely homologous toany bee-genome sequence or human-genome sequence. While it will beappreciated that more than one gene may be targeted in order to maximizethe cytotoxic effect on the Varroa mites, compositions that compriseone, or a few, small RNA's would increase the probability of being aselective insecticide composition as cross reactivity with other insectsmay be reduced.

According to one aspect, a dsRNA composition can be preparedcorresponding to the Varroa destructor Calmodulin-1 and Calmodulin-2genes (e.g. using nucleic acid agents having the sequence as set forthin SEQ ID NOs: 1 to 4, and 69 to 89, their complements or nucleic acidsdirected to regions thereof).

It will be appreciated that as well as down-regulating a number ofgenes, the present disclosure further provides for, and includes, usinga number of agents to down-regulate the same gene (e.g. a number ofnucleic acids, or dsRNAs, each hybridizing to a different segment of thesame gene). For example, in an aspect a combination of one or morenucleic acids corresponding to a sequence selected from the groupconsisting of SEQ ID NOs: 1 to 4, 6, 23, 26 to 35, and 69 to 89 may beused to increase the cytotoxic and anti-parasitic effects of thecomposition. Tools which are capable of identifying species-specificsequences may be used for this purpose—e.g. BLASTN and other suchcomputer programs. U.S. Patent Publication NOs. 20090118214 and20120108497 provide for the use of dsRNA for preventing and treatingviral infections in honeybees. U.S. Patent Publication Nos. 20120258646provides for the use of dsRNA to control Varroa destructor in honeybee.Each publication is hereby incorporated in their entireties.

The present disclosure provides for, and includes, compositions andmethods for down-regulating the expression of a gene in a targetorganism. In an aspect the target organism may be a parasite. In certainaspects, the parasite may be Varroa destructor. As used herein, the term“down-regulating expression” refers to causing, directly or indirectly,reduction in the transcription of a desired gene, reduction in theamount, stability or translatability of transcription products (e.g.RNA) of the gene, and/or reduction in translation of the polypeptide(s)encoded by the desired gene. Down-regulating expression of a geneproduct of a Varroa destructor mite can be monitored, for example, bydirect detection of gene transcripts (for example, by PCR), by detectionof polypeptide(s) encoded by the gene or bee pathogen RNA (for example,by Western blot or immunoprecipitation), by detection of biologicalactivity of polypeptides encode by the gene (for example, catalyticactivity, ligand binding, and the like), or by monitoring changes in theVarroa destructor mite (for example, reduced proliferation of the mite,reduced virulence of the mite, reduced motility of the mite etc) and bytesting bee infectivity/pathogenicity.

Downregulation of a pest or parasite gene product can be effected on thegenomic and/or the transcript level using a variety of agents whichinterfere with transcription and/or translation (e.g., RNA silencingagents, Ribozyme, DNAzyme and antisense nucleic acid molecules).Downregulation of a Varroa destructor mite gene product can be effectedon the genomic and/or the transcript level using a variety of agentswhich interfere with transcription and/or translation (e.g., RNAsilencing agents, Ribozyme, DNAzyme and antisense nucleic acidmolecules).

According to one aspect, the agent which down-regulates expression of apest or parasite gene product is a small RNA, such as an RNA silencingagent. According to this aspect, the small RNA is greater than 15 basepairs in length. In another aspect, the small RNA is greater than 50base pairs in length. In an aspect, the small RNA is greater than 50base pairs in length but less than about 500 base pairs. In an aspect,the small RNA is greater than 100 base pairs in length but less thanabout 500 base pairs. In an aspect, the small RNA is greater than 200base pairs in length but less than about 500 base pairs. In an aspect,the pest or parasite may be a Varroa destructor mite.

Another method of down-regulating a pest or parasite gene product is byintroduction of small inhibitory RNAs (siRNAs). Another method ofdown-regulating a Varroa mite gene product is by introduction of smallinhibitory RNAs (siRNAs).

In one aspect of the present disclosure, synthesis of RNA silencingagents suitable for use with the present disclosure can be effected asfollows. First, the pest or parasite target mRNA is scanned downstreamof the AUG start codon for AA dinucleotide sequences. Occurrence of eachAA and the 3′ adjacent 19 nucleotides is recorded as potential siRNAtarget sites. Preferably, siRNA target sites are selected from the openreading frame, as untranslated regions (UTRs) are richer in regulatoryprotein binding sites. UTR-binding proteins and/or translationinitiation complexes may interfere with binding of the siRNAendonuclease complex (Tuschl ChemBiochem. 2:239-245). It will beappreciated though, that siRNAs directed at untranslated regions mayalso be effective, as demonstrated for GAPDH wherein siRNA directed atthe 5′ UTR mediated about 90% decrease in cellular GAPDH mRNA andcompletely abolished protein level (available on the internet atwww.ambion.com/techlib/tn/91/912.html).

Second, potential target sites are compared to an appropriate genomicdatabase (e.g., human, bee, monarch butterfly, mouse, rat etc.) usingany sequence alignment software, such as the BLAST software availablefrom the NCBI server (available on the internet atwww.ncbi.nlm.nih.gov/BLAST/). Putative target sites which exhibitsignificant homology to other coding sequences are filtered out.

Qualifying target sequences are selected as template for siRNAsynthesis. Preferred sequences are those including low G/C content asthese have proven to be more effective in mediating gene silencing ascompared to those with G/C content higher than 55%. Several target sitesare preferably selected along the length of the target gene or sequencefor evaluation. For better evaluation of the selected siRNAs, a negativecontrol is preferably used in conjunction. Negative control siRNApreferably include the same nucleotide composition as the siRNAs butlack significant homology to the genome. Thus, a scrambled nucleotidesequence of the siRNA is preferably used, provided it does not displayany significant homology to any other gene or pest or parasite targetsequence. An example of a scrambled nucleotide sequence is provided atSEQ ID NO. 5.

For example, a siRNA that may be used in this aspect of the presentdisclosure is one which targets a mite-specific calmodulin gene.Examples of siRNAs are provided in SEQ ID NOs: 3, 4, 88 and 89.

It will be appreciated that the RNA silencing agent of the presentdisclosure need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides.

In some aspects, the RNA silencing agent provided herein can befunctionally associated with a cell-penetrating peptide. As used herein,a “cell-penetrating peptide” is a peptide that comprises a short (about12-residues) amino acid sequence or functional motif that confers theenergy-independent (i.e., non-endocytotic) translocation propertiesassociated with transport of the membrane-permeable complex across theplasma and/or nuclear membranes of a cell. The cell-penetrating peptideused in the membrane-permeable complex of the present disclosurepreferably comprises at least one non-functional cysteine residue, whichis either free or derivatized to form a disulfide link with adouble-stranded ribonucleic acid that has been modified for suchlinkage. Representative amino acid motifs conferring such properties arelisted in U.S. Pat. No. 6,348,185, the contents of which are expresslyincorporated herein by reference. The cell-penetrating peptides of thepresent disclosure preferably include, but are not limited to,penetratin, transportan, pls1, TAT(48-60), pVEC, MTS, and MAP.

Another agent capable of down-regulating a pest or parasite gene productis a DNAzyme molecule capable of specifically cleaving an mRNAtranscript or DNA sequence of the bee pathogen polypeptide. DNAzymes aresingle-stranded polynucleotides which are capable of cleaving bothsingle and double stranded target sequences (Breaker, R. R. and Joyce,G. Chemistry and Biology 1995; 2:655; Santoro, S. W. & Joyce, G. F.Proc. Natl, Acad. Sci. USA 1997; 943:4262) A general model (the “10-23”model) for the DNAzyme has been proposed. “10-23” DNAzymes have acatalytic domain of 15 deoxyribonucleotides, flanked by twosubstrate-recognition domains of seven to nine deoxyribonucleotideseach. This type of DNAzyme can effectively cleave its substrate RNA atpurine:pyrimidine junctions (Santoro, S. W. & Joyce, G. F. Proc. Natl,Acad. Sci. USA 199; for a review of DNAzymes, see Khachigian, L M, CurrOpin Mol Ther 4:119-21 (2002)). In an aspect, the pest or parasite geneproduct may be a Varroa mite gene product. Downregulation of pest orparasite gene products can also be effected by using an antisensepolynucleotide capable of specifically hybridizing with an mRNAtranscript encoding the pest or parasite gene product. Design ofantisense molecules which can be used to efficiently downregulate a pestor parasite gene product must be effected while considering two aspectsimportant to the antisense approach. The first aspect is delivery of theoligonucleotide into the cytoplasm of the appropriate cells, while thesecond aspect is design of an oligonucleotide which specifically bindsthe designated mRNA or RNA target sequence within cells in a way whichinhibits translation thereof. In an aspect, the pest or parasite geneproduct may be a Varroa mite gene product. In another aspect, the pestor parasite gene product may be calmodulin gene product.

A number of delivery strategies which can be used to efficiently deliveroligonucleotides into a wide variety of cell types (see, for example,Luft J Mol Med 76: 75-6 (1998); Kronenwett et al. Blood 91: 852-62(1998); Rajur et al. Bioconjug Chem 8: 935-40 (1997); Lavigne et al.Biochem Biophys Res Commun 237: 566-71 (1997) and Aoki et al. (1997)Biochem Biophys Res Commun 231: 540-5 (1997)).

In addition, algorithms for identifying those sequences with the highestpredicted binding affinity for their target mRNA based on athermodynamic cycle that accounts for the energetics of structuralalterations in both the target mRNA and the oligonucleotide are alsoavailable (see, for example, Walton et al. Biotechnol Bioeng 65: 1-9(1999)). Such algorithms have been successfully used to implement anantisense approach in cells. For example, the algorithm developed byWalton et al. enabled scientists to successfully design antisenseoligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosisfactor-alpha (TNF alpha) transcripts. The same research group has morerecently reported that the antisense activity of rationally selectedoligonucleotides against three model target mRNAs (human lactatedehydrogenase A and B and rat gp1) in cell culture as evaluated by akinetic PCR technique proved effective in almost all cases, includingtests against three different targets in two cell types withphosphodiester and phosphorothioate oligonucleotide chemistries. Inaddition, several approaches for designing and predicting efficiency ofspecific oligonucleotides using an in vitro system were also published(Matveeva et al., Nature Biotechnology 16: 1374-1375 (1998)).

Another agent capable of down-regulating a pest or parasite gene productis a ribozyme molecule capable of specifically cleaving an mRNAtranscript encoding the Varroa mite gene product. Ribozymes are beingincreasingly used for the sequence-specific inhibition of geneexpression by the cleavage of mRNAs encoding proteins of interest (Welchet al., Curr Opin Biotechnol. 9:486-96 (1998)). The possibility ofdesigning ribozymes to cleave any specific target RNA, including viralRNA, has rendered them valuable tools in both basic research andtherapeutic applications. In an aspect, the pest or parasite geneproduct may be a Varroa mite gene product. In another aspect, the pestor parasite gene product may be calmodulin gene product.

An additional method of down-regulating the expression of a pest orparasite gene product in cells is via triplex forming oligonucleotides(TFOs). Recent studies have shown that TFOs can be designed which canrecognize and bind to polypurine/polypyrimidine regions indouble-stranded helical DNA in a sequence-specific manner. Theserecognition rules are outlined by Maher III, L. J., et al., Science(1989) 245:725-7; Moser, H. E., et al., Science, (1987) 238:645-6; Beal,P. A., et al., Science (1992) 251:1360-1363; Cooney, M., et al., Science(1988) 241:456-459; and Hogan, M. E., et al., EP Publication 375408.Modification of the oligonucleotides, such as the introduction ofintercalators and backbone substitutions, and optimization of bindingconditions (pH and cation concentration) have aided in overcominginherent obstacles to TFO activity such as charge repulsion andinstability, and it was recently shown that synthetic oligonucleotidescan be targeted to specific sequences (for a recent review see Seidmanand Glazer, J Clin Invest 2003; 112:487-94). In an aspect, the pest orparasite gene product may be a Varroa mite gene product. In anotheraspect, the pest or parasite gene product may be calmodulin geneproduct.

In general, the triplex-forming oligonucleotide has the sequencecorrespondence:

oligo 3′--A G G T duplex 5′--A G C T duplex 3′--T C G A

However, it has been shown that the A-AT and G-GC triplets have thegreatest triple helical stability (Reither and Jeltsch, BMC Biochem,2002, September 12, Epub). The same authors have demonstrated that TFOsdesigned according to the A-AT and G-GC rule do not form non-specifictriplexes, indicating that the triplex formation is indeed sequencespecific.

Triplex-forming oligonucleotides preferably are at least 15, morepreferably 25, still more preferably or more nucleotides in length, upto 50 or 100 bp.

Transfection of cells (for example, via cationic liposomes) with TFOs,and formation of the triple helical structure with the target DNAinduces steric and functional changes, blocking transcription initiationand elongation, allowing the introduction of desired sequence changes inthe endogenous DNA and resulting in the specific downregulation of geneexpression.

Detailed description of the design, synthesis and administration ofeffective TFOs can be found in U.S. Patent Publication Nos. 2003/017068and 2003/0096980 to Froehler et al., and 2002/0128218 and 2002/0123476to Emanuele et al., and U.S. Pat. No. 5,721,138 to Lawn.

The polynucleotide down-regulating agents of the present disclosure maybe generated according to any polynucleotide synthesis method known inthe art such as enzymatic synthesis or solid phase synthesis. Equipmentand reagents for executing solid-phase synthesis are commerciallyavailable from, for example, Applied Biosystems. Any other means forsuch synthesis may also be employed; the actual synthesis of thepolynucleotides is well within the capabilities of one skilled in theart and can be accomplished via established methodologies as detailedin, for example, “Molecular Cloning: A laboratory Manual” Sambrook etal., (1989); “Current Protocols in Molecular Biology” Volumes I-IIIAusubel, R. M., ed. (1994); Ausubel et al., “Current Protocols inMolecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal,“A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York(1988) and “Oligonucleotide Synthesis” Gait, M. J., ed. (1984) utilizingsolid phase chemistry, e.g. cyanoethyl phosphoramidite followed bydeprotection, desalting and purification by for example, an automatedtrityl-on method or HPLC.

The polynucleotide agents of the present disclosure may compriseheterocylic nucleosides consisting of purines and the pyrimidines bases,bonded in a 3′ to 5′ Sphosphodiester linkage. Preferably usedpolynucleotide agents are those modified in either backbone,internucleoside linkages or bases, as is broadly described hereinunder.

Specific examples of polynucleotide agents useful according to thisaspect of the present disclosure include polynucleotide agentscontaining modified backbones or non-natural internucleoside linkages.Polynucleotide agents having modified backbones include those thatretain a phosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Modified polynucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms can also be used.

Alternatively, modified polynucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,0, S and CH₂ component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;5,602,240; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;5,663,312; 5,214,134; 5,466,677; 5,610,289; 5,633,360; 5,677,437; and5,677,439.

Other polynucleotide agents which can be used according to the presentdisclosure, are those modified in both sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example for such an polynucleotidemimetic, includes peptide nucleic acid (PNA). A PNA polynucleotiderefers to a polynucleotide where the sugar-backbone is replaced with anamide containing backbone, in particular an aminoethylglycine backbone.The bases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. United Statespatents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Other backbone modifications,which can be used in the present disclosure are disclosed in U.S. Pat.No. 6,303,374.

Polynucleotide agents of the present disclosure may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified bases include but are not limited to other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further bases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 613, and those disclosed by Sanghvi, Y. S.,Chapter 15, Antisense Research and Applications, pages 289-2, Crooke, S.T. and Lebleu, B., ed., CRC Press, 1993. Such bases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the disclosure. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi Y S et al. (1993) AntisenseResearch and Applications, CRC Press, Boca Raton 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Following synthesis, the polynucleotide agents of the present disclosuremay optionally be purified. For example, polynucleotides can be purifiedfrom a mixture by extraction with a solvent or resin, precipitation,electrophoresis, chromatography, or a combination thereof.Alternatively, polynucleotides may be used with no, or a minimum of,purification to avoid losses due to sample processing. Thepolynucleotides may be dried for storage or dissolved in an aqueoussolution. The solution may contain buffers or salts to promoteannealing, and/or stabilization of the duplex strands.

It will be appreciated that a polynucleotide agent of the presentdisclosure may be provided per se, or as a nucleic acid constructcomprising a nucleic acid sequence encoding the polynucleotide agent.Typically, the nucleic acid construct comprises a promoter sequencewhich is functional in the host cell, as detailed herein below.

The polynucleotide sequences of the present disclosure, under thecontrol of an operably linked promoter sequence, may further be flankedby additional sequences that advantageously affect its transcriptionand/or the stability of a resulting transcript. Such sequences aregenerally located upstream of the promoter and/or downstream of the 3′end of the expression construct.

The term “operably linked”, as used in reference to a regulatorysequence and a structural nucleotide sequence, means that the regulatorysequence causes regulated expression of the linked structural nucleotidesequence. “Regulatory sequences” or “control elements” refer tonucleotide sequences located upstream, within, or downstream of astructural nucleotide sequence, and which influence the timing and levelor amount of transcription, RNA processing or stability, or translationof the associated structural nucleotide sequence. Regulatory sequencesmay include promoters, translation leader sequences, introns, enhancers,stem-loop structures, repressor binding sequences, terminationsequences, pausing sequences, polyadenylation recognition sequences, andthe like.

It will be appreciated that the nucleic acid agents can be delivered tothe pest or parasite in a great variety of ways. According to oneaspect, the nucleic acid agents are delivered directly to the pest orparasite (e.g. by spraying a mite infested hive). The nucleic acidagents, or constructs encoding same may enter the mites bodies bydiffusion. In this aspect, the promoter of the nucleic acid construct istypically operational in mite cells. In an aspect, the pest or parasitemay be Varroa destructor.

It will be appreciated that since many parasites use their mouths topuncture the host arthropod exoskeleton and feed on the arthropod'shemolymph, the present disclosure contemplates delivering thepolynucleotide agents of the present disclosure to the arthropod,whereby they become presented in the arthropod hemolymph therebybecoming available to the pest or parasite. Thus, according to anotheraspect, the nucleic acid agents are delivered indirectly to the pest orparasite (for example to a mite via a host bee). In this aspect, thepromoter of the nucleic acid construct is typically operational in hostcells. In certain aspects, the pest or parasite may be Varroa destructorand the host arthropod may be a bee.

According to one aspect, the nucleic acid agents are delivered to theinfested hosts by spraying. The nucleic acid agents, or constructsencoding same may enter the host's bodies by diffusion.

In certain aspects, the pest or parasite may be Varroa destructor andthe host arthropod may be a bee.

According to another aspect, the nucleic acid agents are delivered tothe host via its food. The present inventors consider that followingingestion of the nucleic acid agents of the present disclosure, theagents can be presented, for example in a host arthropod in the host'shemolymph, whereby it becomes available to the parasite, for example aVarroa mite.

Thus the polynucleotides of the present disclosure may be synthesized invitro or in vivo, for example in a bacterial or yeast cell, and added tothe food. For example double stranded RNA may be synthesized by addingtwo opposing promoters (e.g. T7 promoters) to the ends of the genesegments, wherein the promoter is placed immediately 5′ to the gene andthe promoter is placed immediately 3′ to the gene segment in theopposite orientation. The dsRNA may then be prepared by transcribing invitro with the T7 RNA polymerase.

Examples of sequences for synthesizing nucleic acids, including dsRNA,according to aspects of the present disclosure are provided in SEQ IDNOs: 1 to 4, 6, 23, 26 to 35, and 69 to 89.

It will be appreciated that some pests or parasites cause wound sites inthe exoskeleton of a host arthropod. Such wound sites harbor bacterialinfections. For example, a host bee wound site may harbor a bacteriasuch as Melissococcus pluton, which causes European foulbrood. Inaddition, to their parasitic effects, parasites are known to act asvectors for a number of other pathogens and parasites. For example,Varroa mites are suspected of acting as vectors for a number of honeybee pathogens, including deformed wing virus (DWV), Kashmir bee virus(KBV), acute bee paralysis virus (ABPV) and black queen cell virus(BQCV), and may weaken the immune systems of their hosts, leaving themvulnerable to infections.

Thus, by killing the pest or parasite (or preventing reproductionthereof), the anti-parasitic, anti-pest or insecticidal agents of thepresent disclosure may be used to prevent and/or treat bacterialinfections of host organisms. For example, Melissococcus pluton andviral infections in host bees caused by the above named viruses. SinceVarroa mite infestation and viral infections are thought to beresponsible for colony collapse disorder (CCD), the present agents mayalso be used to prevent or reduce the susceptibility of a bee colony toCCD.

It will be appreciated that in addition to feeding of anti-parasitic,anti-pest or insecticidal nucleic acid agents for reduction of the beepathogen infection and infestation, enforcement of proper sanitation(for example, refraining from reuse of infested hives) can augment theeffectiveness of treatment and prevention of infections.

Also included and provided for by the present disclosure are transgenicbacteria and yeast cells that express a selective insecticide. In oneaspect, a nucleic acid encoding a small RNA, dsRNA, miRNA or a small ormiRNA-resistant target nucleic acid molecule used herein is operablylinked to a promoter and optionally a terminator. In some embodiments,the transgenic bacteria and yeast cells are killed, for example, byapplying heat or pressure. In some embodiments, the transgenic bacteriaand yeast cells are lysed prior to providing the selective insecticideto the target organism. In some embodiments, the transgenic bacteria andyeast cells are not lysed.

In one aspect, an exogenous nucleic acid molecule used herein is orencodes a small RNA, or in a particular aspect a siRNA, which canmodulate the expression of a gene in a target organism. In an aspect, anexogenous nucleic acid encodes a small RNA having at least 80%, 85%,88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity to asequence selected from the group consisting of SEQ ID NOs: 1-4 and 6-89.In a further aspect, an exogenous nucleic acid molecule used herein isor encodes a dsRNA molecule. In another aspect, an exogenous nucleicacid molecule used herein is or encodes an artificial miRNA. In afurther aspect, an exogenous nucleic acid molecule used herein is orencodes an siRNA. In one aspect, an exogenous nucleic acid molecule usedherein is or encodes a precursor of a small RNA. In another aspect, anexogenous nucleic acid molecule used herein is or encodes a precursor ofa miRNA or siRNA. In one aspect, an exogenous nucleic acid molecule usedherein is a naturally-occurring molecule. In another aspect, anexogenous nucleic acid molecule used herein is a synthetic molecule.

In one aspect, an exogenous nucleic acid molecule used herein is orencodes a stem-loop precursor of a small RNA or in a particular aspect amiRNA, comprising a sequence having at least 80%, 85%, 88%, 90%, 92%,95%, 96%, 97%, 98%, or 99% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 1-4 and 6-89. A stem-loop precursorused herein comprises a sequence having at least 60%, 65%, 70%, 75%,80%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity toa sequence selected from the group consisting of SEQ ID NOs: 1-4 and6-89.

In one aspect, an exogenous nucleic acid molecule used herein is nakedRNA or expressed from a nucleic acid expression construct, where it isoperably linked to a regulatory sequence.

In one aspect, a recombinant DNA construct or a transgene disclosedherein further comprises a transcription terminator.

It is expected that during the life of a patent maturing from thisapplication many relevant methods for down-regulating expression of geneproducts can be developed and the scope of the term “down-regulatingexpression of a gene product of a Varroa destructor mite” is intended toinclude all such new technologies a priori.

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate aspects, may also beprovided in combination in a single aspect. Conversely, various featuresof the disclosure, which are, for brevity, described in the context of asingle aspect, may also be provided separately or in any suitablesubcombination or as suitable in any other described aspect of thedisclosure. Certain features described in the context of various aspectsare not to be considered essential features of those aspects, unless theaspect is inoperative without those elements. Various aspects andaspects of the present disclosure as delineated hereinabove and asclaimed in the claims section below find experimental support in thefollowing examples.

EXAMPLES Example 1 Varroa Mite Calmodulin Gene Sequences

The Calmodulin (CAM) genes provided in Table 1 (SEQ ID NO: 1 and 2), ortheir corresponding transcripts, were used as targets of polynucleotidecompositions comprising a polynucleotide that is at least 18 contiguousnucleotides identical or complementary to those genes or transcripts.The gene sequences provided in Table 1, protein sequences encoded bythose genes, or sequences contained within those genes were used toobtain orthologous Calmodulin (CAM) genes from other arthropod pest andparasitic species not listed in Table 1. Such orthologous genes andtheir transcripts can then serve as targets of polynucleotides providedherein or as a source of anti-parasitic, anti-pest or insecticidalpolynucleotides that are specifically designed to target the orthologousgenes or transcripts.

TABLE 1 Target Calmodulin (CAM) genes of Varroa destructor Gene nameSEQ ID Open reading frame DNA sequence CAM-1 1ATGGCTGATCAGCTAACTGAGGAACAGATCGCCGAGTTCAAAGAGGCGTTTAGCCTGTTTGACAAGGACGGAGATGGCACGATCACGACAAAGGAGCTCGGTACGGTAATGCGATCTCTCGGCCAGAACCCCACTGAGGCTGAACTGCAGGACATGATCAACGAGGTCGACGCCGACGGCTCCGGAACGATAGATTTCCCTGAGTTCCTCACAATGATGGCAAGAAAGATGAAGGACACCGACTCGGAGGAGGAGATCCGAGAGGCGTTCCGCGTATTCGACAAGGATGGCAACGGTTTCATTTCGGCGGCCGAGCTCAGGCACGTTATGACCAACCTTGGCGAGAAGCTTACGGACGAGGAGGTAGATGAGATGATTCGGGAGGCAGATATTGACGGTGATGGTCAGGTCAACTACGAGGAGTTCGTCACCATGATGACGTCCAAGTAA CAM-2 2ATGGCGGATCAGCTGACCGAGGAGCAAATCGCCGAATTCAAGGAGGCTTTCAGCCTGTTCGATAAAGACGGTGATGGCACAATTACGACCAAGGAACTAGGGACCGTCATGCGGTCCCTCGGCCAGAACCCTACTGAGGCTGAGCTTCAAGACATGATCAACGAGGTCGACGCTGACGGTAACGGCACTATTGACTTTCCAGAGTTTCTCACGATGATGGCGCGTAAAATGAAGGACACCGACTCCGAGGAGGAGATCCGGGAAGCTTTTAGGGTTTTTGATAAAGACGGAAATGGCTTCATTTCGGCTGCAGAGCTGAGGCACGTAATGACCAACCTTGGCGAAAAGCTCACGGACGAGGAAGTGGACGAGATGATCCGCGAGGCGGATATCGACGGCGACGGACAGGTCAACTACGAGGAGTTCGTCACGATGATGACATCAAAATGA

For each Calmodulin DNA gene sequence provided in SEQ ID NO: 1 and 2,single stranded or double stranded DNA or RNA fragments in sense orantisense orientation or both are fed in vitro to Varroa mites grown ona petri plate or applied topically to bee hives to effect the expressionof the CAM target genes and obtain a reduction in Varroa destructor mitepopulation.

Example 2 Suppression of Calmodulin (CAM) Genes of Varroa destructor

Polynucleotides for the suppression of expression of Calmodulin (CAM)genes in Varroa destructor mite corresponding to SEQ ID NOs: 3 and 4(Table 2) are provided and were used to used to suppress expression ofCalmodulin (CAM) genes in Varroa destructor mite. The SEQ ID NOs: 3 and4 describe a 373 bp dsRNA polynucleotide sequence and a 186 bp dsRNApolynucleotide sequence, respectively, selected from CAM-1 (SEQ ID NO:1). SEQ ID NO: 3, corresponding to dsRNA polynucleotide CAM_L/CAM373covers most of the open reading frame of the Calmodulin CAM-1 (SEQ IDNO: 1) gene. SEQ ID NO 4, corresponding to dsRNA polynucleotideCAM_S/CAM186 is a partial fragment of CAM_L/CAM373 (SEQ ID NO: 3) and isalso derived from CAM-1 (SEQ ID NO: 1). SEQ ID NO: 5 in Table 2 is acontrol dsRNA sequence polynucleotide sequence with no more than 19 bpsequence identity to any known Varroa destructor gene.

TABLE 2 dsRNAs targeting Varroa destructor Calmodulin (CAM) genesdsRNA name  SEQ ID Nucleic acid sequence CAM L/CAM 3ACAGAUCGCCGAGUUCAAAGAGGCGUUUAGCCUGUUUGACAAGGACGGAGAUGGCACGAUCACGACAAAGGAG373CUCGGUACGGUAAUGCGAUCUCUCGGCCAGAACCCCACUGAGGCUGAACUGCAGGACAUGAUCAACGAGGUCGACGCCGACGGCUCCGGAACGAUAGAUUUCCCUGAGUUCCUCACAAUGAUGGCAAGAAAGAUGAAGGACACCGACUCGGAGGAGGAGAUCCGAGAGGCGUUCCGCGUAUUCGACAAGGAUGGCAACGGUUUCAUUUCGGCGGCCGAGCUCAGGCACGUUAUGACCAACCUUGGCGAGAAGCUUACGGACGAGGAGGUAGAUGAGAUGAUUCGGGAGGCAGAUAUUGAC CAM_S/CAM 4ACAAUGAUGGCAAGAAAGAUGAAGGACACCGACUCGGAGGAGGAGAUCCGAGAGGCGUUCCGCGUAUUCGACA186AGGAUGGCAACGGUUUCAUUUCGGCGGCCGAGCUCAGGCACGUUAUGACCAACCUUGGCGAGAAGCUUACGGACGAGGAGGUAGAUGAGAUGAUUCGGGAGGCAGAUAUUGAC SCRAM 5AUACUUACUGGUGCUAAUUUUUAUCGAGGAUGCCCAACUCCCCCCACUUUAAAACUGCGAUCAUACUAACGAACUCCCGAAGGAGUGAAAGGUGUCUAUGUUGAGCUUAAUAACCUACCUUGCGAGCAAAGAAGGACUAGUUGACCCUGGGCACCCUAUAUUGUUAUGUUGUUUCGAACUGAGUUGGCACCCAUGCUGCACAUGCAACAAACAUGUCGGCCUUCGUGUCUAUCCUAGAAAAGUACCUGUGAACUUGGCUGUCUACAUCAUCAUC

Example 3 Varroa destructor Bioassay at 3 Day Post-Treatment withSpecific dsRNAs

Adult female mites were collected from honeybee colonies and placed in apetri dish plate on top of an artificial diet solution containing amixture of 1% tryptone, 0.5% yeast extract, 1% NaCl and 15 mg/mL agar.In this example the diet was supplemented with 50 μg kanamycin per 1 mLof diet solution. The diet/agar solution was further supplemented with200-500 μg/mL of dsRNA and the resulting solution was poured on a petridish. The dsRNA in this example consisted of either SEQ ID NO: 3(CAM_L/CAM373) or SEQ ID NO: 5 (SCRAM). Fifteen mites were applied toeach plate and the experiment was conducted in triplicate. The dietplates with the mites were incubated at 29° C. with 50-60% relativehumidity. At specific time intervals the plates were inspected and deadmites were counted and removed. For mortality studies the mites werecounted three days after being placed on the diet (FIG. 2). FIG. 2 showsthat all mites were dead at three day after treatment compared tountreated plates or plates where the mites were fed on a dietsupplemented with the non-specific (SCRAM) dsRNA polynucleotide.

Example 4 Varroa destructor Bioassay at 5 Day Post-Treatment with dsRNAsTargeting Calmodulin

Adult female mites were collected from honeybee colonies and placed in apetri dish plate on top of an artificial diet solution. The artificialdiet contained a mixture of 1% tryptone, 0.5% yeast extract, 1% NaCl and15 mg/mL agar. In this example the diet was further supplemented withAntimycotic Solution (100×, Sigma Aldrich) at 8× final concentration,500 μg/mL kanamycin and 220 U/mL nystatin. The diet/agar solution wasfurther supplemented with 200-500 μg/mL of dsRNA and the resultingsolution was poured on a petri dish. The dsRNA in this example consistedof either SEQ ID NO: 3 (CAM_L/CAM373), or SEQ ID NO: 4 (CAM_S/CAM186),or SEQ ID NO: 5 (SCRAM). Fifteen mites were applied to each plate andthe experiment was conducted in triplicate. The diet plates with themites were incubated at 29° C. with 50-60% relative humidity. Atspecific time intervals the plates were inspected and dead mites werecounted and removed. For mortality studies the mites were counted atfive days after being placed on the diet (FIG. 3). For molecularanalysis, live mites were removed from the plates, snap frozen in liquidnitrogen and TAQMAN™ analysis was performed to assess the levels ofCalmodulin (CAM) RNA. FIG. 3, Panel A. the RNA levels for Calmodulin(CAM) genes in mites exposed to SEQ ID NO: 3 (CAM_L/CAM373) or SEQ IDNO: 4 (CAM_S/CAM186) was highly reduced compared to the non-specific(SCRAM) treatment or no treatment (CNTR). FIG. 3, Panel B, astatistically significant mortality in mites that were exposed to dsRNAagainst Calmodulin (CAM) was observed at 5 days after treatment.

Example 5 Method for Delivering of dsRNA Polynucleotides TargetingVarroa Genes Using a Spray-Dried or Semi-Solid Formulation

dsRNA used to suppress expression of Varroa target Calmodulin (CAM)genes was prepared in a formulation containing 1 part dsRNA and ˜14parts trehalose in a phosphate buffer (a solution of 1.15 mM KH₂PO₄(monobasic) and 8 mM Na₂HPO₄ (dibasic), pH 8.0) as illustrated in Table3. Using a Büchi B-290 mini spray dryer, the liquid formulation wasatomized into droplets and heated with gas to produce a flowable powder.

TABLE 3 Formulation Preparation Ratio of AI Stock buffer (X % Finalbuffer (X % Total Stock dsRNA Active Ingre- Active Ingre- (dsRNA) toBuffer w/v trehalose + w/v trehalose + vol buffer stock dient (AI) concdient (AI) conc (trehalose + dsRNA phosphate buffer) phosphate buffer)(mL) (mL) (mL) Ratio (mg/mL) (% solids) phosphate buffer) CAM_L/CAM37340 10 1100 275.00 825.00 ¼ 7.20 0.720 13.9 CAM_S/CAM186 40 10 1285321.21 963.75 ¼ 6.75 0.675 14.8

The resulting particles were formulated with powdered sugar and appliedevenly to hives by spreading the powdered sugar evenly on top of theframes. In other aspects, a semi-solid preparation of the spray-driedmaterial is prepared with water and the sugar-water (“bee-candy”)formulation is fed to the bee hives by allowing the bees to feed on it.

Example 6 In Vivo Reduction of Varroa Mite in Bee Hives after Treatmentwith dsRNA Targeting Calmodulin (CAM) Genes

Varroa mites infesting adult honey bees in the hives were collected andcounted using standard mite counting methodology. Hives were treatedwith spray dried dsRNA according to Example 7 comprising SEQ ID NO: 3(CAM-L), SEQ ID NO: 4 (CAM-S), or no treatment (CONTROL). The mite loadof each hive was assessed at the beginning of the experiment and at 2weeks, 4 weeks and 12 weeks after treatment. FIG. 4 shows the mite loadof the treated hives compared to the hives that did not receive thetreatment. The number of mites counted was normalized to 100 adult beesand is representative of the Varroa mite load.

Example 7 Detection of Transitive Small RNAs in Varroa FollowingTreatment with dsRNA Targeting Calmodulin (CAM) Genes

Varroa mites were collected from hives treated with SEQ ID NO: 3 dsRNApolynucleotides and collected from the hive at 7 day after treatment.Varroa RNA was extracted and small RNA sequencing analysis performedusing the SOLiD platform. The majority of small RNA molecules weredetected outside the dsRNA sequence region and specifically toward the3′ portion of the dsRNA region of SEQ ID NO: 3. Additionally, themajority of the transitive reads were in the antisense orientationrelative to the Calmodulin (CAM) gene transcript sequence. Further,small RNAs specific for CAM-2 (SEQ ID NO: 2) were detected in thisexperiment despite the hives being treated with dsRNA for SEQ ID NO: 3,which is predicted to be specific for CAM-1 (SEQ ID NO: 1). Thisobservation supports the hypothesis that suppression of RNA expressionand transitive small RNA generation in Varroa works even when only asmall fragment between the two genes shares complete identity at the DNAlevel (in this case 23 nucleotides).

Example 8 Calmodulin (CAM) Gene Homologs from Arthropod Pest andParasite Species and Corresponding dsRNA Polynucleotides

Using standard bioinformatics technique and the sequences SEQ ID NOs: 1and 2 for Varroa destructor a set of 31 conserved Calmodulin (CAM) genesequences were identified in arthropod pest species that infest eitherother arthropods or mammals and that will be targeted for generegulation. These sequences were identified and presented as aphylogenetic tree in FIG. 1. The DNA sequences in FIG. 1 were furtheranalyzed by identifying the conserved 373 bp domain within each sequencethat corresponds to SEQ ID NO: 3 (CAM_L/CAM373). Table 4 lists the SEQID NOs of the newly identified Calmodulin (CAM) gene sequences as wellas the corresponding 373 bp dsRNA polynucleotide trigger sequences. The373 bp polynucleotide dsRNA sequences will be tested either alone or incombination in direct feeding assays against their respective arthropodspecies.

TABLE 4 Calmodulin (CAM) gene sequences identified from arthropod pestsor parasites and their corresponding 373 bp RNA polynucleotides. SEQ IDNO Gene Name Organism/Species Type 6 CAM-3 Varroa destructor cDNA 7CAM-1 Ixodes scapularis cDNA 8 CAM-1 Aedes aegypti cDNA 9 CAM-1 Culexquinquefasciatus cDNA 10 CAM-1 Acyrthosiphon pisum cDNA 11 CAM-1Harpegnathos saltator cDNA 12 CAM-1 Pediculus humanus cDNA corporis 13CAM-1 Anopheles gambiae cDNA 14 CAM-1 Solenopsis invicta cDNA 15 CAM-1Ixodes scapularis RNA 16 CAM-1 Aedes aegypti RNA 17 CAM-1 Culexquinquefasciatus RNA 18 CAM-1 Acyrthosiphon pisum RNA 19 CAM-1Harpegnathos saltator RNA 20 CAM-1 Pediculus humanus RNA corporis 21CAM-1 Anopheles gambiae RNA 22 CAM-1 Solenopsis invicta RNA 23 CAM-3Varroa destructor RNA 24 CAM-1 Tetranychus urticae cDNA 25 CAM-1Tetranychus urticae RNA 26 CAM-4 Varroa destructor cDNA 27 CAM-4 Varroadestructor RNA 28 CAM-5 Varroa destructor cDNA 29 CAM-5 Varroadestructor RNA 30 CAM-7 Varroa destructor cDNA 31 CAM-7 Varroadestructor RNA 32 CAM-8 Varroa destructor cDNA 33 CAM-8 Varroadestructor RNA 34 CAM-9 Varroa destructor cDNA 35 CAM-9 Varroadestructor RNA 36 CAM Ixodes scapularis cDNA 37 CAM Ixodes scapularisRNA 38 CAM Ixodes scapularis cDNA 39 CAM Ixodes scapularis RNA 40 CAMIxodes scapularis cDNA 41 CAM Ixodes scapularis cDNA 42 CAM Ixodesscapularis RNA 43 CAM Aedes aegypti cDNA 44 CAM Aedes aegypti RNA 45 CAMAedes aegypti cDNA 46 CAM Aedes aegypti RNA 47 CAM Aedes aegypti cDNA 48CAM Aedes aegypti RNA 49 CAM Culex quinquefasciatus cDNA 50 CAM Culexquinquefasciatus RNA 51 CAM Culex quinquefasciatus cDNA 52 CAM Culexquinquefasciatus RNA 53 CAM Culex quinquefasciatus cDNA 54 CAM Culexquinquefasciatus RNA 55 CAM Culex quinquefasciatus cDNA 56 CAM Culexquinquefasciatus RNA 57 CAM Acyrthosiphon pisum cDNA 58 CAMAcyrthosiphon pisum RNA 59 CAM Acyrthosiphon pisum cDNA 60 CAMAcyrthosiphon pisum RNA 61 CAM Pediculus humanus cDNA 62 CAM Pediculushumanus RNA 63 CAM Pediculus humanus cDNA 64 CAM Pediculus humanus RNA65 CAM Pediculus humanus cDNA 66 CAM Pediculus humanus RNA 67 CAMPediculus humanus cDNA 68 CAM Pediculus humanus RNA

Example 9 Varroa Calmodulin (CAM) Gene Transcripts and dsRNA TriggerSequences

The Calmodulin (CAM) sequences provided in Table 5 (SEQ ID NOs: 69 and70), or their corresponding transcripts, were used as targets ofpolynucleotide compositions comprising a polynucleotide that is at least18 contiguous nucleotides identical or complementary to those genes ortranscripts. The 5′ and 3′UTR sequences for the Varroa Calmodulinsequences were identified by RNA sequencing.

TABLE 5 Target transcripts for Calmodulin (CAM) genes of Varroadestructor Gene name and Species SEQ ID NO Type CAM-1; Varroa destructor69 RNA CAM-2; Varroa destructor 70 RNA

SEQ ID NOs: 69 and 70 were tiled in 150 bp fragments. Table 6illustrates the top strand (5′-3′) for the 150 bp fragments that tileacross SEQ ID NOs: 69 and 70.

TABLE 6 Tiled polynucleotide sequences for CAM-1 and CAM-2 genes SEQ IDPosition within Gene name NO transcript sequence CAM-1 71  1-150 CAM-172 151-300 CAM-1 73 301-450 CAM-1 74 451-600 CAM-1 75 601-750 CAM-1 76751-900 CAM-1 77  901-1050 CAM-1 78 1051-1200 CAM-1 79 1201-1350 CAM-180 1351-1500 CAM-2 81  1-150 CAM-2 82 151-300 CAM-2 83 301-450 CAM-2 84451-600 CAM-2 85 601-750 CAM-2 86 751-900 CAM-2 87  901-1050

One or more dsRNA comprising a sequence selected from SEQ ID NOs: 71-87is provided in vitro to Varroa mites grown on a petri plate or appliedtopically to bee hives to effect the expression of the CAM target genesand obtain a reduction in Varroa destructor mite population.

Example 10 In Vitro Bioassay of Calmodulin (CAM) Targeting Triggers inVarroa Mite

Polynucleotide trigger sequences targeting Calmodulin (CAM)-1 and 2 weregenerated based on conserved sequence overlap between CAM-1 and CAM-2sequences. These are presented as SEQ ID NOs: 88 and 89 (targeting CAM-1and CAM-2, respectively).

Polynucleotide sequences selected from SEQ ID NOs: 88 and 89 were testedin an in vitro bioassay for their ability to suppress viability of adultVarroa mites. Adult female mites were collected from honeybee coloniesand placed in a petri dish plate on top of an artificial diet solution.The artificial diet contained a mixture of 1% tryptone, 0.5% yeastextract, 1% NaCl and 15 mg/mL agar. In this example, the diet wasfurther supplemented with Antimycotic Solution (100×, Sigma Aldrich) at8× final concentration, 500 μg/mL kanamycin and 220 U/mL nystatin. Thediet/agar solution was further supplemented with 200-500 μg/mL of dsRNAand the resulting solution was poured on a petri dish. The dsRNA in thisexample consisted of either SEQ ID NO: 3 (CAM373), SEQ ID NO: 88(CAM-1), or SEQ ID NO: 89 (CAM-2) or non-treated control (NTC). Fifteenmites were applied to each plate and the experiment was conducted intriplicate. The diet plates with the mites were incubated at 29° C. with50-60% relative humidity. At specific time intervals the plates wereinspected and dead mites were counted and removed. For mortality studiesthe mites were counted at five and six days after being placed on thediet (FIG. 5). Additionally, the dsRNA for SEQ ID NO: 88 (CAM-1) and SEQID NO: 89 (CAM-2) were mixed in equimolar amount and fed as describedabove to the mites. FIG. 6 shows the result of this application.

For molecular analysis, live mites are removed from the plates, snapfrozen in liquid nitrogen and TAQMAN™ analysis is performed to assessthe levels of Calmodulin (CAM) RNA.

Example 11 In Vivo Field Reduction of Varroa Mite Infestation in FieldTreated Bee Hives after Treatment with dsRNA Targeting Calmodulin (CAM)Gene

dsRNA used to suppress expression of Varroa targeted Calmodulin (CAM)genes was prepared by mixing dsRNA stock in Phosphate Buffer with 66%sugar syrup. The liquid formulation was supplied as a syrup to the bees,allowed to feed on it until fully consumes (approximately 2-3 days).Each field testing group consisted of 33 hives. The groups consisted ofnon-treated hives, non-specific trigger treated (SEQ ID NO: 5) andspecific trigger treated (SEQ ID NO: 3). Bees were treated in tworounds, each round consisted of two feedings two weeks apart: at thestart of the delivery (week 0) and two weeks later (week 2), then againon week 13 and 15. Assessment of bee survival was done at 4, 9, 13, 15and 17 weeks (FIG. 7). Significant suppression of Varroa population wasobserved following treatment with the specific trigger (SEQ ID NO:3) atweek 9.

What is claimed is:
 1. A bee-ingestible, bee-absorbable, mite-ingestible, or mite-absorbable composition comprising an excipient and a nucleic acid molecule having a sequence that is at least 96% identical or complementary to at least 21 contiguous nucleotides of SEQ ID NO:
 3. 2. The composition of claim 1, wherein said nucleic acid sequence is a dsRNA.
 3. The composition of claim 1, wherein said excipient is selected from the group consisting of protein, pollen, carbohydrate, polymer, liquid solvent, sugar syrup, sugar solid, and semi-solid feed.
 4. The composition of claim 2, wherein said dsRNA is an siRNA.
 5. The composition of claim 1, wherein said nucleic acid sequence comprises at least 23 contiguous nucleotides of SEQ ID NO:
 3. 6. The composition of claim 2, wherein said dsRNA sequence comprises at least 23 contiguous nucleotides of SEQ ID NO:
 3. 7. A nucleic acid construct comprising a nucleic acid that encodes a sequence that is at least 95% identical or complementary to SEQ ID NO: 3 operably linked to a promoter sequence functional in a host cell and capable of producing a dsRNA when introduced into said host cell.
 8. The nucleic acid construct of claim 7, further comprising at least one regulatory element selected from the group consisting of translation leader sequences, introns, enhancers, stem-loop structures, repressor binding sequences, termination sequences, pausing sequences, and polyadenylation recognition sequences.
 9. The nucleic acid construct of claim 7, wherein said host cell is selected from the group consisting of a bacteria cell and a yeast cell.
 10. A method of providing a composition to a honeybee, comprising providing the bee an effective amount of the composition of claim 1, whereby the nucleic acid is present in honeybee tissue.
 11. The method of claim 10, wherein said calmodulin gene sequence is a Varroa destructor calmodulin gene sequence.
 12. The method of claim 10, wherein said honeybee is a forager or a hive bee.
 13. The method of claim 11, wherein said honeybee is a bee of a colony and said providing reduces the susceptibility of said bee colony to Varroa destructor, reduces the parasitation of said bee colony by Varroa destructor, or reduce the parasite load of said bee colony by Varroa destructor.
 14. The method of claim 11, wherein said providing reduces the susceptibility of said honeybee to Varroa destructor, reduces the parasitation of said honeybee by Varroa destructor, or reduce the parasite load of said honeybee by Varroa destructor.
 15. The composition of claim 1, further comprising one or more nucleic acid molecules having a second nucleic acid sequence that is essentially identical or essentially complementary to a different region of a calmodulin gene sequence. 